Immunogenic compositions and diagnostic and therapeutic uses thereof

ABSTRACT

The present invention relates to a method of inducing an immune response to a parasite utilizing an immunogenic composition comprising a glycosylphosphatidylinositol (“GPI”) inositolglycan domain or its derivative or equivalent. The present invention is useful as a prophylactic and/or therapeutic treatment for microorganism infections of mammals such as parasite infections and particularly infection by  Plasmodium  species. The invention also provides a method of monitoring, or qualitatively or quantitatively assessing an immune response to a microorganism such as a parasite. More particularly, this aspect of the present invention is directed to assessing said immune response utilizing a GPI inositoglycan domain or its derivative or equivalent, which facilitates the qualitative and/or quantitative analysis of anti-GPI antibodies in a biological sample, the identification of unique specificities of antibodies, epitope specific screening or the rational design of immunogenic molecules and the generation, thereby, of functionally effective immunointeractive molecules.

Cross-Reference to Related Application:

This application is the §371 national phase of International ApplicationPCT/AU2003000944, filed Jul. 25, 2003, which claims the benefit of U.S.Provisional Application No. 60/398,607, filed Jul. 26, 2002.

FIELD OF TH INVENTION

The present invention relates generally to a method of eliciting orotherwise inducing an immune response to a microorganism andcompositions for use therein. More particularly, the present inventionrelates to a method of inducing an immune response to a parasiteutilising an immunogenic composition comprising aglycosylphosphatidylinositol (referred to herein as “GPI”)inositolglycan domain or its derivative or equivalent. The presentinvention is useful, inter alia, as a prophylactic and/or therapeutictreatment for microorganism infections of mammals such as, for example,parasite infections and in particular infection by Plasmodium species.

In another aspect the invention provides a method of diagnosing,monitoring, screening for or otherwise qualitatively or quantitativelyassessing an immune response to a microorganism and, in particular, aparasite. More particularly, this aspect of the present invention isdirected to assessing said immune response utilising a GPI inositoglycandomain or its derivative or equivalent. The development of this aspectof the present invention facilitates, inter alia, the qualitative and/orquantitative analysis of anti-GPI antibodies in a biological sample, theidentification and/or isolation of unique specificities of antibodies(such as those which bind a parasite derived toxin or the parasiteitself), epitope specific screening or the rational design ofimmunogenic molecules and the generation, thereby, of functionallyeffective immunointeractive molecules.

BACKGROUND OF TH INVENTION

Bibliographic details of the publications referred to by author in thisspecification are collected at the end of the description.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge in Australia.

The malaria parasite is considered to be one of the single most seriousinfectious agents in the world, infecting 5% of the global populationand causing serious mortality and morbidity to sensitive populations andhampering socio-economic development.

Severe malaria infection shares several clinical features in common withbacterial septic shock. In both conditions, the excess production bymacrophages of pro-inflammatory cytokines such as Tumor Necrosis Factor(TNF), Interleukin-1 (IL-1) and IL-6 occurs in response to malaria“toxins” and lipopolysaccharide (LPS), respectively, leading tocomplications such as fever and hyperpyrexia, leukopenia,thrombocytopenia, hypotension, disseminated intravascular coagulation,leukocyte infiltration, vascular permeability and multi-organinflammation, which may lead eventually to death. Thus, many signs,symptoms and syndromes in acute and severe malaria infection result fromthe activity of a parasite “toxin” released into the circulation duringthe blood-stage developmental cycle of the infection.

GPI has been identified as a candidate toxin of parasite origin(Schofield, L. and Hackett, F. (1993) Journal of Experimental Medicine177:145-153 and Tachado, S. D., Gerold, P., Schwarz, R., Novakovic, S.,McConville, M., and Schofield, L. (1997) Proceedings of the NationalAcademy of Sciences USA 94:4022-4027). The structure of the molecule hasbeen elucidated (Gerold, P., Dieckman-Schuppert A. and Schwarz, R. T.(1992) Bio. Soc. Trans. 29:297 and Gerold, P., Schofield, L., Brackman,M., Holder, A. A., Schwarz, R. T. (1996) Mol. Biochem. Parasitol 75:131)and it comprises a lipidic domain and a glycan domain. Intact GPI occursin two closely related forms, Pfg1α(NH—CH₂—CH₂—PO₄-(Manα1-2)-6Manα1-2Manα1-6Manα1-4GlcN-H₂α1-6(myristoyl)-myo-Inositol-1-PO₄-dipalmitoylglycerol),and Pfg1β(NH—CH₂—CH₂—PO₄-6Manα1-2Manα1-6Manα1,4GlcN-H₂α1-6(myristoyl)-myo-Inositol-1-PO₄-dipalmitoylglycerol).

The parasite derived GPI molecule regulates host cell function and geneexpression in various tissues by activating endogenous GPI-based signaltransduction pathways, involving hydrolysis into second messengers andthe activation of both tyrosine and serine/threonine kinases. This leadsto the activation of the NFκB/c-rel family of transcription factors,which regulate the expression of numerous pro-inflammatory lociimplicated in malarial pathology, such as TNF, IL-1, iNOS and ICAM-1.

The toxin theory of malarial pathogenesis can be ascribed to CamilloGolgi, in 1886, who hypothesized that the proximal cause of the febrileparoxysm was a released toxin of parasite origin (Golgi, C. (1886) Arch.Sci. Med. (Torino) 10:109). Clark proposed that the systemicinflammation of malaria infection resulted from a functional malarialendotoxin, and suggested that this agent exerts systemic effects largelythrough the induction of endogenous pyrogens of host origin. Clarkcorrectly identified TNF as a major host mediator of disease (Clark, I.A. (1978) Lancet 2:75 and Clark, I. A., Virelizier, J.-L., Carswell, E.A., and Wood, P. R. (1981) Infect. Immun. 32:1058). Consequently, theproduction of this and related pyrogenic cytokines (IL-1, IL-6) frommonocyte/macrophages is often taken as a useful surrogate marker for theinitiation of pathological processes in malaria infection. John Playfairand his colleagues extended this work to show that crude extracts ofrodent malaria parasites could induce macrophages to secrete TNF invitro (Bate, C. A., Taverne, J., and Playfair, J. H. (1988) Immunology64:227 and Bate, C. A., Taverne, J., and Playfair, J. H. (1989)Immunology 66:600) and inferred that the toxin included a phospholipidmoiety. Nonetheless, prior to the advent of the present invention, thespecific biochemical identity of the parasite toxin, and its mechanismof action, have remained obscure.

In work leading up to the present invention, the inventors investigatedthe use of portions of GPI to induce protective immunity againstmalarial pathology. The inventors have surprisingly discovered that GPIportions which exclude the lipidic domain induce protective immunitywhereas portions carrying the lipidic domain do not.

In still another surprising aspect, it has been determined that theabove-described GPI portions can be effectively utilised to facilitatethe qualitative and/or quantitative analysis of the immune responsewhich has been generated to a parasite, in particular Plasmodium, toparasite pathology, such as malarial pathology. Still further, theanalysis of the immune response in this unique and surprisinglyeffective antigen-based manner provides a means of facilitating epitopespecific screening or the rational design of functionally effectiveimmunogenic molecules. This aspect of the present invention has beenparticularly facilitated by the successful generation of synthetic GPImolecules.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers.

One aspect of the present invention is directed to a method of elicitingor inducing, in a mammal, an immune response directed to a microorganismsaid method comprising administering to said mammal an effective amountof an immunogenic composition which composition comprises a moleculecapable of inducing an immune response directed to the inositolglycandomain of a GPI but which molecule is substantially incapable ofinducing an immune response directed to a lipidic domain of said GPI.

Another aspect of the present invention provides a method of elicitingor inducing, in a mammal, an immune response directed to a microorganismsaid method comprising administering to said mammal an effective amountof an immunogenic composition which composition comprises a modified GPImolecule or derivative or equivalent thereof and which modified GPImolecule comprises insufficient lipidic domain to induce or elicit animmune response directed to a GPI lipidic domain.

Still another aspect of the present invention is directed to a method ofeliciting or inducing, in a mammal, an immune response directed to aparasite said method comprising administering to said mammal aneffective amount of an immunogenic composition which compositioncomprises the inositolglycan domain portion of a parasite GPI orderivative or equivalent thereof and which inositolglycan domain portioncomprises insufficient lipidic domain of said parasite GPI to induce orelicit an immune response directed to said lipidic domain.

Still yet another aspect of the present invention contemplates a methodof eliciting or inducing, in a mammal, an immune response directed to P.falciparum said method comprising administering to said mammal aneffective amount of an immunogenic composition which compositioncomprises a GPI inositolglycan domain wherein said GPI inositolglycandomain comprises the structure

ethanolamine-phosphate-(Manα1,2)-Manα1,2Manα1,6Manα1,4GlcN-myo-inositolphosphoglycerol

or derivative or equivalent thereof.

Still yet another aspect of the present invention contemplates a methodof eliciting or inducing, in a mammal, an immune response directed to P.falciparum said method comprising administering to said mammal aneffective amount of an immunogenic composition which compositioncomprises a GPI inositolglycan domain wherein said GPI inositolglycandomain comprises the structure

X₁—X₂—X₃—X₄-ethanolamine-phosphate-Manα1,2)-Manα1,2Manα1,6Manα1,4GlcN-myo-inositolphosphoglycerol

wherein X₁, X₂, X₃ and X₄ are any 4 amino acids, or derivative orequivalent of said GPI inositolglycan domain.

Still yet another aspect of the present invention contemplates a methodof eliciting or inducing, in a mammal, an immune response directed to P.falciparum said method comprising administering to said mammal aneffective amount of an immunogenic composition which compositioncomprises a GPI inositolglycan domain wherein said GPI inositolglycandomain comprises the structure

EtN-P-[Mα2]Mα2 Mα6 Mα4Gα6Ino

EtN-P-[Mα2][G]Mα2 Mα6 Mα4Gα6Ino

EtN-P-[Mα2][X]Mα2 Mα6 Mα4Gα6Ino

EtN-P-[Mα2][EtN-P]Mα2 Mα6 Mα4Gα6Ino

EtN-P-Mα2 Mα6 Mα4G

EtN-P-Mα2 Mα6 M

EtN-P-[Mα2][G]Mα2 Mα6 Mα4G

EtN-P-[Mα2][X]Mα2 Mα6 Mα4G

EtN-P-[Mα2][EtN-P]Mα2 Mα6 Mα4G

Mα2 [Mα2][G]Mα2 Mα6 Mα4G

Mα2 [Mα2][X]Mα2 Mα6 Mα4G

Mα2 [Mα2][EtN-P]Mα6 Mα4G

Mα6 Mα4Gα6Ino

Mα2 Mα6 Mα4Gα6Ino

Mα2 [Mα2]Mα6 Mα4Gα6Ino

Mα2 [Mα2][G]Mα6 Mα4Gα6Ino

Mα2 [Mα2][X]Mα2 Mα4Gα6Ino

EtN-P-[Mα2][G]Mα2 Mα6 M

EtN-P-[Mα2][X]Mα2 Mα6 M

EtN-P-[Mα2][EtN-P]Mα2 Mα6 M

Mα2 [Mα2][G]Mα2 Mα6 M

Mα2 [Mα2][X]Mα2 Mα6 M

Mα2 [Mα2][EtN-P]Mα6 M

Mα2 Mα6M

Mα6 Mα4G

EtN-P-[Mα2][G]Mα2 M

EtN-P-[Mα2][X]Mα2 M

EtN-P-[Mα2][EtN-P]Mα2 M

EtN-P-(Manα1,2)-6Mα1, 2Mα1, 6Manα1, 4GlcNH₂α1-myo-inositol-1,2cyclic-phosphate NH₂—CH₂—CH₂—PO₄-(Manα1-2) 6Manα1-2, Manα1-6, Manα1-4GlcNH₂-6myo-inositol-1,2 cyclic-phosphate

or derivative or equivalent thereof wherein EtN is ethanolamine, P isphosphate, M is mannose, G is non-N-acetylated glucosamine, [G] is anynon-N-acetylated hexosamine, Ino is inositol orinositol-phosphoglycerol, [X] is any other substituent, α representsα-linkages which may be substituted with β-linkages wherever required,and numeric values represent positional linkages which may besubstituted with any other positional linkages as required.

A further aspect of the present invention contemplates a method oftherapeutically or prophylactically treating a mammal for amicroorganism infection said method comprising administering to saidmammal an effective amount of an immunogenic composition whichcomposition comprises a molecule capable of inducing an immune responsedirected to the inositolglycan domain of a GPI, but substantiallyincapable of inducing an immune response directed to the lipid domain ofa GPI, for a time and under conditions sufficient for said immuneresponse to reduce, inhibit or otherwise alleviate any one or moresymptoms associated with infection of said mammal by said microorganism.

Another further aspect of the present invention is directed to a methodof therapeutically or prophylactically treating a mammal for amicroorganism infection said method comprising administering to saidmammal an effective amount of an immunogenic composition whichcomposition comprises a modified GPI molecule or derivative orequivalent thereof and which modified GPI molecule comprisesinsufficient lipidic domain to induce or elicit an immune responsedirected to a GPI lipidic domain for a time and under conditionssufficient for said immune response to reduce, inhibit or otherwisealleviate any one or more symptoms associated with infection of saidmammal by said microorganism.

In a related aspect, the present invention provides a method for thetreatment and/or prophylaxis of a mammalian disease conditioncharacterised by a microorganism infection, said method comprisingadministering to said mammal an effective amount of an immunogeniccomposition which composition comprises a molecule capable of inducingan immune response directed to the inositolglycan domain of a GPI, butsubstantially incapable of inducing an immune response directed to thelipid domain of a GPI, for a time and under conditions sufficient forsaid immune response to reduce, inhibit or otherwise alleviate any oneor more symptoms associated with said microorganism infection.

Still another further aspect of the present invention is directed to amethod for the treatment and/or prophylaxis of a mammalian diseasecondition characterised by a microorganism infection said methodcomprising administering to said mammal an effective amount of animmunogenic composition which composition comprises a modified GPImolecule or derivative or equivalent thereof and which modified GPImolecule comprises insufficient lipidic domain to induce or elicit animmune response directed to a GPI lipidic domain for a time and underconditions sufficient for said immune response to reduce, inhibit orotherwise alleviate any one or more symptoms associated with saidmicroorganism infection.

Still yet another aspect of the present invention relates to the use ofa composition comprising a molecule capable of inducing an immuneresponse directed to a microorganism GPI inositolglycan domain butsubstantially incapable of inducing an immune response directed to alipidic domain of GPI in the manufacture of a medicament for thetherapeutic and/or prophylactic treatment of a mammalian diseasecondition characterised by infection with said microorganism.

Still yet another hither aspect of the present invention relates to theuse of an immunogenic composition comprising a Plasmodium GPIinositolglycan domain or derivative or equivalent thereof whichinositolglycan domain comprises insufficient lipidic domain of aPlasmodium GPI to elicit or induce an immune response directed to a GPIlipidic domain in the manufacture of a medicament for the therapeuticand/or prophylactic treatment of a mammalian disease conditioncharacterized by infection with said Plasmodium.

Another aspect of the present invention is directed to a compositioncapable of inducing an immune response directed to a microorganism, saidcomposition comprising a molecule capable of inducing an immune responseagainst a microorganism GPI inositolglycan domain but substantiallyincapable of inducing an immune response to a lipidic domain of a GPI.

Still another aspect of the present invention is directed to acomposition capable of inducing an immune response directed to amicroorganism said composition comprising a modified GPI molecule orderivative or equivalent thereof which modified GPI molecule comprisesinsufficient lipidic domain to induce or elicit an immune responsedirected to a GPI lipidic domain.

Another aspect of the present invention relates to a method ofinhibiting, halting or delaying the onset of progression of a mammaliandisease condition characterized by a microorganism infection said methodcomprising administering to said mammal an effective amount of anantibody has hereinbefore described.

Still yet another aspect of the present invention relates to a vaccinecomposition comprising as the active component a modified GPI moleculeor derivative or equivalent thereof which modified GPI molecule orderivative or equivalent thereof which modified GPI molecule comprisesinsufficient lipidic domain to induce or elicit an immune responsedirected to a GPI lipidic domain.

Still another aspect of the present invention is directed to apharmaceutical composition comprising a molecule capable of inducing animmune response directed to a microorganism GPI inositolglycan domainbut substantially incapable of inducing an immune response directed to alipidic domain of a GPI, as broadly described above, together with oneor more pharmaceutically acceptable carriers and/or diluents.

A further aspect of the present invention is directed to antibodies toGPI inositolglycan domains but substantially incapable of interactingwith the lipidic domain of a GPI.

Yet another aspect of the present invention relates to a pharmaceuticalcomposition comprising an antibody directed to a GPI inositolglycandomain together with one or more pharmaceutically acceptable carriers ordiluents as hereinbefore described.

A further aspect of the present invention relates to the use of theantibodies of the present invention in relation to disease conditions.For example, the present invention is particularly useful but in no waylimited to use in treating parasitic infections, their symptoms andpathologies.

Another aspect of the present invention relates to a method ofinhibiting, halting or delaying the onset of progression of a mammaliandisease condition characterised by a microorganism infection said methodcomprising administering to said mammal an effective amount of anantibody has hereinbefore described.

Accordingly, another aspect of the present invention provides a methodfor detecting, in a biological sample, an immunointeractive moleculedirected to a microorganism said method comprising contacting saidbiological sample with a molecule comprising said microorganism GPIinositolglycan domain or a derivative or equivalent thereof andqualitatively and/or quantitatively screening for said GPIinositolglycan domain-immunointeractive molecule complex formation.

In a related aspect, the present invention provides a method fordetecting, monitoring or otherwise assessing an immune response directedto a microorganism in a subject said method comprising contacting abiological sample, from said subject, with a molecule comprising saidmicroorganism GPI inositolglycan domain or a derivative or equivalentthereof and qualitatively and/or quantitatively screening for GPIinositolglycan domain-immunointeractive molecule complex formation.

In one aspect, the present invention therefore more preferably providesa method for detecting, in a biological sample, an immunointeractivemolecule directed to Plasmodium said method comprising contacting saidbiological sample with the inositolglycan domain portion of a PlasmodiumGPI or derivative or equivalent thereof and qualitatively and/orquantitatively screening for GPI inositolglycan domain-immunointeractivemolecule complex formation.

In a related aspect, the present invention more preferably provides amethod for detecting, monitoring or otherwise assessing an immuneresponse directed to Plasmodium in a subject said method comprisingcontacting a biological sample, from said subject, with theinositolglycan domain portion of a Plasmodium GPI or derivative orequivalent thereof and qualitatively and/or quantitatively screening forGPI inositolglycan domain-immunointeractive molecule complex formation.

One aspect of the present invention therefore most preferably provides amethod for detecting, in a biological sample, an antibody directed toPlasmodium said method comprising contacting said biological sample withthe inositolglycan domain portion of a Plasmodium GPI or a derivative orequivalent thereof and qualitatively and/or quantitatively screening forGPI inositolglycan domain-antibody complex formation.

A related aspect of the present invention most preferably provides amethod for detecting, monitoring, or otherwise assessing an immuneresponse directed to Plasmodium in a subject said method comprisingcontacting a biological sample, from said subject, with theinositolglycan domain portion of a Plasmodium GPI or a derivative orequivalent thereof and qualitatively and/or quantitatively screening forGPI inositolglycan domain-antibody complex formation.

Accordingly, another aspect of the present invention is directed to amodular kit comprising one or more members wherein at least one memberis a solid support comprising a GPI molecule as hereinbefore defined.

Accordingly, the present invention should also be understood to extendto a method for analyzing, designing and/or modifying an agent capableof interacting with an anti-GPI glycan immunointeractive moleculebinding site, which immunointeractive molecule is identifiable utilizingthe diagnostic methodology hereinbefore disclosed, said methodcomprising contacting said immunointeractive molecule or derivativethereof with a putative agent and assessing the degree of interactivecomplementarity of said agent with said binding site.

The present invention also extends to the use of the molecules generatedin accordance with this aspect of the present invention in accordancewith the therapeutic, prophylactic and diagnostic methods hereinbeforedescribed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of the epitope specificity ofanti-GPI antibodies determined by competition ELISA. Sera from miceimmunized with free GPI were screened for reactivity to malarial GPI inthe presence or absence of defined competitors (Phosphatidylinositol orphosphatidylserine).

FIG. 2 is a graphical representation of the results of C57B1/6 miceimmunized with free GPI in IFA and sham-immunized mice (IFA alone) whichwere challenged with P. berghei ANKA and survival assessed over 15 days.

FIG. 3 is a graphical representation of the epitope mapping ofanti-lipid monoclonal antibodies. Monoclonal antibody 1C7 to GPI derivedfrom mice immunized with free GPI were screened by competition ELISA forreactivity with GPI in the presence or absence of PI and GPI glycancompetitors.

FIG. 4 is a graphical representation of monoclonal antibody 1C7, tomalarial GP lipid domains, recognition of mammalian GPIs at the cellsurface as determined by FACS analysis. Solid line, binding of 1C7 tomacrophages; dotted line, no antibody; dashed line, binding of 1C7following PI-PLC treatment of macrophages.

FIG. 5 is a photographic representation of monoclonal 1C7, to lipiddomain of the GPI, induction of rapid onset tyrosylphosphorylation inhost cells.

FIG. 6 is a graphical representation of monoclonal 1C7 synergize withGPI, phorbol esters and parasite extracts in the induction of TNF outputfrom murine C3H/HeJ macrophages.

FIG. 7 is a graphical representation of monoclonal 1C7 exacerbation ofthe P. berghei ANKA cerebral malaria syndrome in C57Bl/6 mice.

FIG. 8 is a graphical representation of polyclonal antisera from miceimmunized with the purified P. falciparum GPI glycan covalentlyconjugated to a protein carrier inhibiting TNF output from macrophagesin response to GPI or total parasite extracts. Values show absorbance at450 mM by anti-TNF ELISA (Pharmingen) and are proportional to mass TNF.

FIG. 9 is a graphical representation of immunization of C57Bl/6 micewith the purified P. falciparum GPI glycan covalently coupled to KLHproviding a significant level of protection against the cerebral malariasyndrome induced by P. berghei ANKA.

FIG. 10 is a graphical representation of GPI being the dominantTNF-inducing toxin of P. falciparum. Monoclonal antibodies 1G7 and 3G6specific for the GPI glycan derived from OVA-TCR mice immunized withOVA-glycan inhibiting TNF output from macrophages in response to totalparasite extracts.

FIG. 11 is a graphical representation of monoclonal antibodies to the P.falciparum GPI inositolglycan, upon passive transfer, substantiallyprotecting mice against cerebral malaria.

FIG. 12 is a graphical representation of monoclonal antibodies to the P.falciparum inositolglycan, upon passive transfer, protecting miceagainst parasite-induced lethal toxic shock.

FIG. 13 is a schematic representation of the synthesis of glycan. 1.Reagents: a. 4, AgOTf, NIS, CH₂Cl₂/Et₂O (38% a); b. NaOMe, CH₂Cl₂/MeOH(83%); c. 6, TMSOTf, CH₂Cl₂ (75%); d. NaOMe, CH₂Cl₂/MeOH (71%); e. 7,TMSOTf, CH₂Cl₂ (92%); f. NaOMe (69%); g. 8, TBSOTf, CH₂Cl₂ (98%); h.NaOMe (83%); i 9, TMSOTF, CH₂Cl₂ (84%); j. (CH₂OH)₂, CSA, CH₃CN (81%);k. Cl₂P(O)OMe, Pyr. (88%); l, TBAF, THF (61%); m, 11, tetrazole, CH₃CN;n. t-BuOOH, CH₃CN (84%, 2 steps); o. DBU, CH₂Cl₂; P. Na, NH₃, THF (75%,2 steps). (AgOTf, silver trifluoromethanesulfonate; NIS,N-iodosuccinimide; CH₂Cl₂, dichloromethane, Et₂O, diethyl ether; NaOMe,sodium methoxide; MeOH, methanol; TMEOTf, trimethylsilyltrifluoromethanesulfonate; TBSOTf, tert-butyldimethylsilyl trifluoromethanesulfonate;CSA, camphorsulfonic acid; CH₃CN, acetonitrile; Cbz, carbobenzyloxy;Pyr, pyridine; TBAF, tetrabutylammonium fluoride; THF, tetrahydrofuran;DBU, 1,8-diazabicyclo[5,4,0]undec-7-ene; Obn, O-benzyl).

FIG. 14 is an image of antibodies raised against synthetic GPI glycanrecognizing native GPI and neutralize toxin activity in vitro. a. Leftpanel, reactivity of anti-glycan IgG antibodies with P. falciparumtrophozoites and schizonts and lack of reactivity to uninfectederythrocytes detected by immunofluorescence assay. Right panel, the samefield under white light illumination. b. Left panel, Western blot ofanti-glycan IgG antibodies (1/200) against parasite-infected (lane 1)and uninfected erythrocytes (lane 2). Right panel, comparison ofreactivity against parasites by two sera from KLH-glycan-immunized mice(lanes 3,4), pre-immune sera from lane 3 donor (lane 5) and shamKLH-immunized mouse (lane 6). All sera were used at 1/400 dilution. Thedetection antibody was peroxidase-conjugated goat anti-mouse IgG(γ-chain specific). DF, dye front. c. Levels of TNFα in culturesupernatants of RAW264.7 cells exposed to medium alone (open square),parasites alone (triangle), or parasites in the presence of variousdilutions of sera from pre-immune (closed circle), sham-immunized(closed square) or glycan immunized mice (open circles).

FIG. 15 is an image of immunization against the synthetic GPI glycansubstantially protecting against murine cerebral malaria, pulmonaryoedema and acidosis. a. Kaplan-Meier survival plots, and b.parasitaemias, to 15 days post infection, of KLH-glycan-immunized(closed circles) and sham-immunized (open squares) mice challenged withP. berghei ANKA. c. Haemotoxylin-Eosin stained sections of brain tissueshowing blood vessels from KLH-glycan immunized (left and center panels)and sham-immunized (right panel) mice sacrificed on day 6post-infection. d. As an index of pulmonary oedema, the ratio of weweight to dry weight of lungs from KLH-glycan-immunized andsham-immunized animals at day 6 post-infection are expressed as aproportion of the lung wet:dry weight ratio of age/sex matcheduninfected controls. e. pH of serum drawn at day 6 from uninfected andP. berghei-ANKA-infected immunized and sham-immunized donors. *, p>0.05.

FIG. 16 is a schematic representation of the synthetic glycan core unit.

FIG. 17 is a schematic representation of the method used to conjugatethe glycan to a carrier protein. Sham conjugation procedures were alsofollowed substituting cysteine for glycan.

FIG. 18 is a representation of the conjugation ratios obtained with thevarious carrier proteins Ovalbumin (OVA), KeyHole Limpet Haemocyanin(KLH) and Bovine Serum Albumin (BSA). Conjugation efficiency wasdetermined by GC/MS analysis of myo-inositol content of protein-glycanconjugate.

FIG. 19 is a graphical representation of the IgG response to syntheticglycan PNG sera 93. Specifically, this figure shows an ELISA test usinghuman serum from a Papua New Guinean donor naturally exposed to malariainfection, reacted against a range of antigens including No Ag (Noantigen at all), BSA-Cys (Sham conjugated BSA-Cysteine), BSA-GLY (BSAconjugated to synthetic GPI glycan) and PfAg (total P. falciparummalaria antigen. The data show that the synthetic GPI glycan is ablespecifically to detect anti-Glycan antibodies in human serum from anindividual exposed to malaria.

FIG. 20 is a graphical representation of the IgG response to syntheticglycan PNG and Melbourne individuals. Specifically, this figure showsthat individuals from a malaria endemic part of Papua New Guinea haveconsiderably higher titres than non-malaria exposed Melbourne donorswhen the synthetic glycan is used as a capture antigen.

FIG. 21 is both a graphical and tabulated representation of competitionELISAS performed on synthetic glycan vs GPI. Specifically, this figureshows that synthetic GPI glycan is able to compete out the majority ofhuman antibody responses to the native GPI toxin when used in molarexcess as a competitor. This defines the fact that the syntheticmaterial is an authentic antigenic match for the native material andalso shows that the majority of anti-toxin serological reactivity isencompassed within the glycan region. These findings are at odds withothers who claim that the majority of human anti-GPI serologicalreactivity is directed towards the lipidic domain (for example Naik, R.S. et al. (2000) Glycosylphosphatidylinositol anchors of Plasmodiumfalciparum: molecular characterization and naturally elicited antibodyresponse that may provide immunity to malaria pathogenesis. J. Exp. Med.192, 1563-1576).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, in part, on the surprisingdetermination that a Plasmodium GPI molecule which excludes the lipidicportion will induce protective immunity whereas a GPI molecule whichcomprises the lipidic domain will not. This determination hasfacilitated the development of compositions and methodology forapplication, inter alia, in the prophylactic or therapeutic treatment ofmicroorganism infection.

It has still further been surprisingly determined that the subject GPImolecule, in particular the synthetic GPI molecule herein disclosed,facilitates a highly effective and informative antigen-based analysis ofone or more qualitative and/or quantitative aspects of the immuneresponse to a parasite, per se, or parasite pathology. The enablement ofthese analyses further facilitate, inter alia, the identification and/orisolation of unique specificities of antibodies, epitope specificscreening or the rational design of immunogenic molecules and thegeneration of functionally effective immunointeractive molecules.

Accordingly, one aspect of the present invention is directed to a methodof eliciting or inducing, in a mammal, an immune response directed to amicroorganism said method comprising administering to said mammal aneffective amount of an immunogenic composition which compositioncomprises a molecule capable of inducing an immune response directed tothe inositolglycan domain of a GPI but which molecule is substantiallyincapable of inducing an immune response directed to a lipidic domain ofsaid GPI.

The present invention is predicated on the surprising observation thatmice immunized with purified, intact, free GPI mount an IgM dominatedresponse directed predominantly to the lipidic domain of the molecule,which cross reacts with host GPI lipidic domains which are exposed athost cell surfaces. The antibodies are not protective clinically againstsubsequent parasite infection. In fact, passive transfer of theseantibodies exacerbates disease severity. However, immunization with theglycan domain of malarial GPI results in IgG antibodies interactive withthe glycan domain of GPI and mice thus immunized are substantiallyprotected against pathology induced by subsequent malaria challenge.Passive transfer of these IgG antibodies is protective againstpathology. The inventors have demonstrated, therefore, that IgMantibodies to the lipidic domain and IgG antibodies to the glycan domainof the malaria GPI differ in their effects, the former promoting diseaseand the latter preventing it. It should be understood that in preventingor minimizing the induction of an immune response directed to the GPI ofa microorganism, the onset of an immune response directed to lipidicdomain of the subject mammal (host) is thereby prevented or minimized byvirtue of minimizing the production of antibodies to a microorganism GPIwhich would otherwise cross-react with the host GPI.

GPIs are ubiquitous among eukaryotes; described from T. brucei, T.cruzi, Plasmodium, Leishmania, and Toxoplasma, as well as yeast, insect,fish and numerous mammalian sources (for recent reviews see McConville,M. J. and Ferguson, M. A., (1993) Biochem. J. 294:305 and Stevens, V. L.(1995) Biochem. J. 310:361). GPIs consist of a conserved core glycan(Manα1-2Manα1-6Manα1-4GlcNH₂) linked to the 6-position of themyo-inositol ring of phosphatidylinositol (PI). GPIs are built up on thecytoplasmic face of the endoplasmic reticulum (ER) by the sequentialaddition of sugar residues to PI by the action of glycosyltransferases.The maturing GPI is then translocated across the membrane to the luminalside of the ER, whence it may be exported to the cell surface, free orin covalent association with proteins. The tetrasaccharide core glycanmay be further substituted with sugars, phosphates and ethanolaminegroups in a species and tissue-specific manner. GPI fatty acid moietiescan be either diacylglycerols, alkylacylglycerols, monoalkylglycerols orceramides, with additional palmitoylations or myristoylations to theinositol ring. The overall picture is of a closely related family ofglycolipids sharing certain core features but with a high level ofvariation in fatty acid composition and side-chain modifications to theconserved core glycan.

Accordingly, reference herein to “GPI inositolglycan domains” should beread as including reference to all forms of GPI inositolglycan domainsand derivatives or equivalents thereof. The term “GPI inositolglycan” isused interchangeably with terms such as but not limited to“inositolglycan” (IG), “inositophosphoglycan” (IPG),“phosphoinositolglycan” (PIG), “phosphooligosaccharide” (POS) and themolecules described by these terms should be understood as “GPIinositolglycan” molecules. It should also be understood that referenceto “GPI inositolglycan domain” includes reference to a GPIinositolglycan domain linked, bound or otherwise associated withnon-inositolglycan molecules such as, but not limited to, the glycerollinker sequence which links the lipidic domain to the inositolglycandomain, a non-immunogenic portion of the lipidic domain or an amino acidpeptide.

Preferably the molecule is a portion of GPI which comprises theinositolglycan domain or derivative or equivalent thereof butsubstantially does not contain a portion capable of inducing an immuneresponse directed to a lipidic domain of said GPI.

Accordingly, the present invention more particularly provides a methodof eliciting or inducing, in a mammal, an immune response directed to amicroorganism said method comprising administering to said mammal aneffective amount of an immunogenic composition which compositioncomprises a modified GPI molecule or derivative or equivalent thereofand which modified GPI molecule comprises insufficient lipidic domain toinduce or elicit an immune response directed to a GPI lipidic domain.

Preferably, said modified GPI molecule is the inositolglycan domainportion of GPI or derivative or equivalent thereof.

Still without limiting the present invention in any way, theadministration of an immunogenic composition comprising aninositolglycan domain portion of GPI or derivative or equivalent thereofsubstantially lacking the lipidic domain, as hereinbefore defined, isalso thought to benefit the subject mammal by minimizing certainunwanted responses which may otherwise occur incidentally to immuneresponse induction, but which enhance disease severity, if the subjectimmunogenic molecule comprised a lipid domain. Specifically;

-   (i) the intact GPI is a toxin and may induce non-immunological    physiological sensitization in recipients such that the response to    the natural GPI toxin is exacerbated upon malaria challenge. The    inventors have shown that the lipidic portion of the intact GPI is    necessary for full toxic activity by virtue of its' ability to    initiate lipid-dependent signaling in host cells, and act as a    lipidic second messenger;-   (ii) intact glycolipids may associate with host CD1 molecules for    presentation to CD1-restricted NKT cells or other unusual T cell    lineages. These T cells are known to produce high levels of    cytokines such as interferon-γ and IL-4 very rapidly in response to    stimulation and are likely to be crucial regulators of downstream    TH1/TH2 differentiation. Immunization with purified, intact (i.e.    lipidated), free GPI may result in priming of these T cells which    subsequently respond with high levels of interferon-γ upon parasite    challenge, thereby exacerbating the disease syndromes. That is,    immunological sensitization of unusual T cells may contribute to the    phenomenon of exacerbated disease severity.

“Derivatives” and “equivalents” should be understood to includefragments, parts, portions, chemical equivalents, mutants, homologs andanalogs. Chemical equivalents of a GPI inositolglycan domain can act asa functional analog of the GPI inositolglycan domain. For example, achemical equivalent of the GPI inositolglycan domain includes a GPIinositolglycan domain in which the phosphoglycerol component of theinositolglycan has been modified to increase hydrophobicity. This may beachieved by replacement with truncated, partial or modified fatty acidsor other hydrophobic moieties and acts to improve the immunogenicity orstability of the molecule, without generating an undesirable antibodyresponse. In another example, a chemical equivalent includes GPI glycanin which the terminal inositol-phosphoglycerol is replaced withinositol-1,2 cyclic-phosphate. Without limiting the present invention inany way, such a change will not substantially alter the functionalproperties of the derivatised GPI glycan relative to non-derivatisedmolecules. Rather, such a substitution is the inherent outcome ofcertain chemical synthesis procedures. Chemical equivalents may notnecessarily be derived from a GPI inositolglycan domain but may sharecertain confirmational similarities. Alternatively, chemical equivalentsmay be specifically designed to mimic certain immunological andphysiochemical properties of the GPI inositolglycan domain. Chemicalequivalents may be chemically synthesized or may be detected following,for example, natural product screening. Chemical equivalents alsoinclude synthetic carbohydrates and peptide mimics. Homologs of GPIinositolglycan domains contemplated herein include, but are not limitedto, GPI inositolglycan domains from different species including, forexample, Saccharomyces. Fragments, include portions such as the glycancomponent of the inositolglycan domain, which portions are effective inachieving the object of the present invention.

GPI inositolglycan domains suitable for use in the present invention maybe derived from any natural, recombinant or synthetic source. Thisincludes, for example, GPI inositolglycan domains derived by geneticmanipulation of expression systems, and by manipulations of the GPIpost-translational modifications of proteins via recombinant DNAtechniques such as glycosylation inhibitors. It also includes chemicallysynthetic or semi-synthetic inositolglycan domains and fragments thereofderived by any chemical process including the use of enzymes for theaddition or removal of residues.

The term “microorganism” should be understood in its broadest sense andincludes, for example, the parasitic and fungal taxa Plasmodium,Trypanosoma, Leishmania, Toxoplasma and Candida. “Microorganism” shouldalso be understood to extend to molecules which are secreted by or shedfrom the subject organism. This would include for example, toxinmolecules or molecules which are cleared from the surface of themicroorganism. Preferably, the GPI inositolglycan domain suitable foruse in the present invention is a parasite GPI inositolglycan domain andeven more preferably a Plasmodium GPI inositolglycan domain.

Accordingly, the present invention is preferably directed to a method ofeliciting or inducing, in a mammal, an immune response directed to aparasite said method comprising administering to said mammal aneffective amount of an immunogenic composition which compositioncomprises the inositolglycan domain portion of a parasite GPI orderivative or equivalent thereof and which inositolglycan domain portioncomprises insufficient lipidic domain of said parasite GPI to induce orelicit an immune response directed to said lipidic domain.

Even more preferably said parasite GPI inositolglycan domain is aPlasmodium GPI inositolglycan domain or derivative or equivalentthereof.

Most preferably, said Plasmodium is P. falciparum.

Yet even more preferably, the present invention contemplates a method ofeliciting or inducing, in a mammal, an immune response directed to P.falciparum said method comprising administering to said mammal aneffective amount of an immunogenic composition which compositioncomprises a GPI inositolglycan domain wherein said GPI inositolglycandomain comprises the structure

ethanolamine-phosphate-(Manα1,2)-Manα1,2Manα1,6Manα1,4GlcN-myo-inositolphosphoglycerol

or derivative or equivalent thereof.

In another most preferred embodiment the immunogenic compositioncomprises a GPI inositolglycan domain wherein said GPI inositolglycandomain comprises the structure

X₁—X₂—X₃—X₄-ethanolamine-phosphate-(Manα1,2)-Manα1,2Manα1,6Manα1,4GlcN-myo-inositolphosphoglycerol

wherein X₁, X₂, X₃ and X₄ are any 4 amino acids, or derivative orequivalent of said GPI inositolglycan domain.

In still another preferred embodiment the immunogenic compositioncomprises a GPI inositolglycan domain wherein said GPI inositolglycandomain comprises a structure selected from:

EtN-P-[Mα2]Mα2 Mα6 Mα4Gα6Ino

EtN-P-[Mα2][G]Mα2 Mα6 Mα4Gα6Ino

EtN-P-[Mα2][X]Mα2 Mα6 Mα4Gα6Ino

EtN-P-[Mα2][EtN-P]Mα2 Mα6 Mα4Gα6Ino

EtN-P-Mα2 Mα6 Mα4G

Mα2 Mα6 Mα4G

EtN-P-Mα2 Mα6 M

EtN-P-[Mα2][G]Mα2 Mα6 Mα4G

EtN-P-[Mα2][X]Mα2 Mα6 Mα4G

EtN-P-[Mα2][EtN-P]Mα2 Mα6 Mα4G

Mα2 [Mα2][G]Mα2 Mα6 Mα4G

Mα2 [Mα2][X]Mα2 Mα6 Mα4G

Mα2 [Mα2][EtN-P]Mα6 Mα4G

Mα6 Mα4Gα6Ino

Mα2 Mα6 Mα4Gα6Ino

Mα2 [Mα2]Mα6 Mα4Gα6Ino

Mα2 [Mα2][G]Mα6 Mα4Gα6Ino

Mα2 [Mα2][X]Mα6 Mα4Gα6Ino

EtN-P-[Mα2][G]Mα2 Mα6 M

EtN-P-[Mα2][X]Mα2 Mα6 M

EtN-P-[Mα2][EtN-P]Mα2 Mα6 M

Mα2 [Mα2][G]Mα2 Mα6 M

Mα2 [Mα2][X]Mα2 Mα6 M

Mα2 [Mα2][EtN-P]Mα6 M

Mα2 Mα6 M

Mα6 Mα4G

EtN-P-[42][G]Mα2 M

EtN-P-[Mα2][X]Mα2 M

EtN-P-[Mα2][EtN-P]Mα2 M

or derivative or equivalent thereof wherein EtN is ethanolamine, P isphosphate, M is mannose, G is non-N-acetylated glucosamine, [G] is anynon-N-acetylated hexosamine, Ino is inositol orinositol-phosphoglycerol, [X] is any other substituent, α representsα-linkages which may be substituted with β-linkages wherever required,and numeric values represent positional linkages which may besubstituted with any other positional linkages as required.

Any of these structures may be further modified by substituents ofpositive, negative or neutral charge such as phosphates,phosphoglycerol, hexosamines, amino acids, thiols etc in any positionand with any type of linkage. These structures may be further modifiedby addition of any number of amino acids for the purpose of providing alinkage sequence.

Reference to “derivative” herein should be understood to encompass, inone preferred embodiment, an immunogenic composition comprising a GPIinositolglycan domain derivative wherein the terminalinositol-phosphoglycerol is substituted with inositol-1,2cyclic-phosphate. Without limiting the present invention in any way,such a substitution is a characteristic outcome where certain forms ofchemical synthesis are utilized, such as that exemplified in Example 18.

Accordingly, in yet still another preferred embodiment, the immunogeniccomposition comprises a GPI inositolglycan domain wherein said GPIinositolglycan domain comprises the structure

EtN-P-(Manα1,2)-6Mα1, 2Mα1, 6Manα1, 4GlcNH₂α1-myo-inositol-1,2cyclic-phosphate

or derivative or equivalent thereof wherein EtN is ethanolamine, P isphosphate and M is mannose.

Even more preferably, the immunogenic composition comprises a GPIinositolglycan domain wherein said GPI inositolglycan domain comprisesthe structure

NH₂—CH₂—CH₂—PO₄-(Manα1-2) 6Manα1-2Manα1-6Manα1-4GlcNH₂-6myo-inositol-1,2 cyclic-phosphate

or derivative or equivalent thereof.

It should be understood that non-N-acetylated hexosamine includesglucosamine or any other nitrous-acid labile substituent. It should befurther understood that any of these structures may be further modifiedby substituents including, but not limited to, of positive, negative orneutral charge such as phosphates, phosphoglycerol, hexosamines, aminoacids or thiols in any position and with any type of linkage.

The GPI inositolglycan domain of the present invention may be conjugatedto another molecule. Said conjugation may be performed for any one ormore reasons such as, but not limited to:

-   (i) The GPI inositolglycan domain may be too small to be antigenic.    Accordingly, conjugation to a carrier molecule, such as a protein,    may be required such that said GPI inositolglycan domain, which    forms part of the GPI inositolglycan domain-conjugate, acts as a    hapten and immunity is induced to said GPI inositolglycan domain.    The carrier protein may be selected from a range of antigenic    proteins such as but not limited to recombinant proteins derived    from Plasmodium gene sequences, tetanus toxoid, purified protein    derivative, hepatitis B or Key Hole Limpet Haemocyanin and Diptheria    toxoid.-   (ii) The GPI inositolglycan domain when conjugated with specific    anti-pathogen vaccine molecules (such as anti-malarial vaccine    molecules) may result in the production of anti-inositolglycan    domain antibodies which reverse the immune suppression that    otherwise may occur in response to exposure to the native form of    the vaccine molecule where said molecule is itself GPI-anchored. For    example, the GPI inositolglycan domain may be coupled to a malarial    recombinant protein which can act as both a carrier protein and a    vaccine in its own right.

Without intending to limit this aspect of the present invention to anyone theory or mode of action, primary and secondary T lymphocyteresponses to some GPI-anchored surface protein antigens are inhibited bythe GPI anchor. Examples of such protein antigens includesCircumsporozoite (CS) proteins of P. falciparum and P. berghei and themembrane-form of Variant Surface Glycoprotein of F. brucei. Sinceimmunization against synthetic or recombinant peptides or proteins ofGPI-anchored surface molecules such as the CS protein, MSP-1, MSP-2 orMSP-4, for example, may be insufficient to allow MHC Class II anamnesticboosting when the native antigens are encountered during naturalparasitic challenge due to the induction of immunosuppression,immunization against the GPI moiety provides a means to alleviate thisimmunosuppression.

-   (iii) The GPI inositolglycan domain may comprise only part of the    target epitope. For example, peptide sequences, other carbohydrates    (and any associated post-translational modifications) corresponding    to C-terminal domains of native GPI-anchored proteins or    GPI-anchored glycoconjugates may also form part of the target GPI    inositolglycan domain epitope. Removal of any part of the epitope    (by removing the portion of the C-terminal domain which forms part    of the GPI inositolglycan domain epitope) may lead to reduction or    loss of binding of antibodies. Said peptide sequences or    carbohydrates would therefore be conjugated to said GPI    inositolglycan domain. For example, some antibodies to malarial GPI,    while specifically neutralizing GPI function, recognise epitopes    which predominantly include the inositolglycan but also include    portions of the protein to which the GPIs are actually bound in    nature, i.e. the adjacent C-terminal portions of GPI-anchored    proteins. The presence of peptide domains can also improve the    affinity of certain antibodies, for example by helping to stabilise    the inositolglycan conformationally. Furthermore, such conjugation    can render a relatively unimmunogenic inositolglycan domain    sufficiently immunogenic. Specifically, the inclusion of a    C-terminal peptide determinant, for example, may help increase the    immunogenicity of the inositolglycan by forming a composite antigen    which is more immunologically foreign than inositolglycan alone.

The resulting GPI inositolglycan domain-conjugate may be administered asa preparation formulated in or with an adjuvant. The adjuvant isselected from the range of adjuvants known to induce high levels ofantibody, including water in oil emulsions, oil in water emulsions,water in oil in water double emulsions, saponin, Quil A extracts andother derivatives of saponin, DEAE-dextran, dextran sulphate, aluminiumsalts and nonionic block co-polymers. The adjuvant may include otherimmunomodulators, such as muramyl-dipeptide and derivatives, cytokines,and cell wall components from species of mycobacteria or corynebacteria.The adjuvant formulation may include a combination of two or more of theadjuvants listed. These lists are not to be taken as exhaustive. Theselection of adjuvant is in part dependent on the species being targetedand is based on the level and duration of the immune response requiredand on the lack of reactogenicity (ie tissue compatibility). The levelof active component and adjuvant are chosen to achieve the desired leveland duration of immune response.

Host GPIs play a significant role in the normal physiological regulationof various cellular responses in higher eukaryotes. Foreign GPIs such asGPIs of parasite origin exert pathophysiological effects, andspecifically regulate host cell function, by acting as a mimic ofendogenous host GPI signalling pathways. Signal transduction induced inhost cells by GPI's of P. falciparum, T. brucei, and L. mexicana, forexample, activate the macrophage lineage-specific hck member of thesrc-family of protein tyrosine kinases within 30 seconds of addition tocells (Tachado et al (1997), supra). Protein tyrosine kinase (PTK)activation is required for downstream gene expression resulting inphosphorylation, cell signalling and TNF, IL-1, iNOS, ICAM-1 and VCAMexpression (Schofield, L., Novakovic, S., Gerold, P., Schwarz, R. T.,McConville, M. J. and Tachado S. D. (1996) J. Immunol. 156:1886-1896,Tachado, S. D., Gerold, P., McConville, M. J., Baldwin, T., Quilici, D.,Schwarz, R. T., and Schofield, L. (1996) Journal of Immunology156:1897-1907 and Tachado et al (1997), supra). PTK activation maps tothe inositolglycan moiety of GPI and follows binding of the core glycanto a receptor on the surface of cells (Tachado et al (1997), supra).Parasite GPIs appear to activate similar kinases as those activated uponperturbation of endogenous GPI-anchored proteins at the cell surface.

The toxic nature of foreign GPIs such as parasite GPIs can beexemplified with respect to malarial GPIs. When inoculated in vivo, themalarial GPI induces pyrexia and symptoms of acute malaria and causesthe death of the recipient in a standard assay of TNF driven lethality(Schofield and Hackett (1993), supra). In addition to inducing TNF andIL-1 expression in macrophages, the GPI exerts several other TNFindependent effects on host tissues that may contribute to pathologicalprocesses in malaria infections. GPI directly increases expression ofE-selectin, ICAM and VCAM in vascular endothelial cells (Schofield et al(1996), supra). GPI also induces de novo protein synthesis of inducablenitric oxide synthase and the production of NO in a time and dosedependent manner, from macrophages and synergises with interferon-γ inthis activity (Tachado et al (1996) supra). In the hypoxic or ischaemicmodel, cerebral malaria is proposed to result from a blockage of thepost capillary venules of the brain by sequestered parasite infectederythrocytes binding to the adhesion molecules ICAM, VCAM and E-selectin(Berendt. A. R., Turner, G. D. H. and Newbold, C. I. (1994) ParasitolToday 10:412, 1994). GPI can therefore be lethal in vivo and inducemalarial symptomology encompassing both systemic inflammation andorgan-specific pathology such as the cerebral syndrome.

Foreign GPIs may also induce immunosuppression. GPIs isolated from P.falciparum and T. brucei, for example, when added at low concentrationsto cultures of CD4+ and CD8+ α/β TCR+ T cells block cell cycleprogression and cellular proliferation, inhibiting the upregulation ofIL-2 R/CD25 and CD28 expression and blocking expression of IL-2,interferon γ, and IL-4. The GPIs also inhibit the T cell proliferativeresponse to IL-2. In vivo, GPI anchored surface proteins such as malariaCS protein, MSP-1, MSP-2, and the membrane form variant surfaceglycoprotein of T. brucei inhibit, via the covalently associated GPIanchor, primary and secondary T lymphocyte responses to said antigens.

While not intending to limit the present invention to any one theory ormode of action, immunisation with a GPI molecule lacking the lipiddomain induces an IgG response to the inositolglycan domain which blockssubsequent parasitic GPI action. Both toxicity and immunosuppression, asdescribed above, are significantly reduced.

A further aspect of the present invention relates to the use of theinvention in relation to disease conditions. For example, the presentinvention is particularly useful, but in no way limited to use intherapeutically or prophylactically treating parasitic infections suchas by immunizing a mammal against a parasitic infection. In this regard,it should be understood that the method of the present invention isdirected to inducing an immune response for the purpose of alleviatingor preventing the onset of symptoms associated with a parasiticinfection (such as toxicity and immunosuppression) and/or where the GPIdomain is conjugated to a suitable antipathogen molecule, reducing orpreventing parasitic infection. Reference herein to “symptoms”associated with a microorganism infection should be understood to extendto both the infection itself as well as the physical and/orphysiological consequences (such as toxicity or immunosuppression) ofsuch an infection.

Accordingly, another aspect of the present invention contemplates amethod of therapeutically or prophylactically treating a mammal for amicroorganism infection said method comprising administering to saidmammal an effective amount of an immunogenic composition whichcomposition comprises a molecule capable of inducing an immune responsedirected to the inositolglycan domain of a GPI, but substantiallyincapable of inducing an immune response directed to the lipid domain ofa GPI, for a time and under conditions sufficient for said immuneresponse to reduce, inhibit or otherwise alleviate any one or moresymptoms associated with infection of said mammal by said microorganism.

More particularly, the present invention is directed to a method oftherapeutically or prophylactically treating a mammal for amicroorganism infection said method comprising administering to saidmammal an effective amount of an immunogenic composition whichcomposition comprises a modified GPI molecule or derivative orequivalent thereof and which modified GPI molecule comprisesinsufficient lipidic domain to induce or elicit an immune responsedirected to a GPI lipidic domain for a time and under conditionssufficient for said immune response to reduce, inhibit or otherwisealleviate any one or more symptoms associated with infection of saidmammal by said microorganism.

Preferably, said microorganism is a parasite and even more preferablyPlasmodium falciparum.

In accordance with this preferred aspect of the present invention, theimmunogenic composition preferably comprises a GPI inositolglycan domainwherein said GPI inositolglycan domain comprises the structure

ethanolamine-phosphate-(Manα1,2)-Manα1,2Manα1,6Manα1,4GlcN-myo-inositolphosphoglycerol

or derivative or equivalent thereof.

In another preferred embodiment, the subject inositolglycan domaincomprises the structure

X₁—X₂—X₃—X₄-ethanolamine-phosphate-(Manα1,2)-Manα1,2Manα1,6Manα1,4GlcN-myo-inositolphosphoglycerol

wherein X₁, X₂, X₃ and X₄ are any 4 amino acids, or derivative orequivalent of said GPI inositolglycan domain.

In still another preferred embodiment, the subject inositolglycan domaincomprises a structure selected from:

EtN-P-[Mα2]Mα2 Mα6 Mα4Gα6Ino

EtN-P-[Mα2][G]Mα2 Mα6 Mα4Gα6Ino

EtN-P-[Mα2][X]Mα2 Mα6 Mα4Gα6Ino

EtN-P-[Mα2][EtN-P]Mα2 Mα6 Mα4Gα6Ino

EtN-P-Mα2 Mα6 Mα4G

Mα2 Mα6 Mα4G

EtN-P-Mα2 Mα6 M

EtN-P-[Mα2][G]Mα2 Mα6 Mα4G

EtN-P-[Mα2][X]Mα2 Mα6 Mα4G

EtN-P-[Mα2][EtN-P]Mα2 Mα6 Mα4G

Mα2 [Mα2][G]Mα2 Mα6 Mα4G

Mα2 [Mα2][X]Mα2 Mα6 Mα4G

Mα2 [Mα2][EtN-P]Mα6 Mα4G

Mα6 Mα4Gα6Ino

Mα2 Mα6 Mα4Gα6Ino

Mα2 [Mα2]Mα2 Mα4Gα6Ino

Mα2 [Mα2][G]Mα6 Mα4Gα6Ino

Mα2 [Mα2][X]Mα6 Mα4Gα6Ino

EtN-P-[Mα2][G]Mα2 Mα6 M

EtN-P-[Mα2][X]Mα2 Mα6 M

EtN-P-[Mα2][EtN-P]Mα2 Mα6 M

Mα2 [Mα2][G]Mα2 Mα6 M

Mα2 [Mα2][X]Mα2 Mα6 M

Mα2 [Mα2][EtN-P]Mα6 M

Mα2 Mα6 M

Mα6 Mα4G

EtN-P-[Mα2][G]Mα2 M

EtN-P-[Mα2][X]Mα2 M

EtN-P-[Mα2][EtN-P]Mα2 M

or derivative or equivalent thereof wherein EtN is ethanolamine, P isphosphate, M is mannose, G is non-N-acetylated glucosamine, [G] is anynon-N-acetylated hexosamine, Ino is inositol orinositol-phosphoglycerol, [X] is any other substituent, α representsα-linkages which may be substituted with β-linkages wherever required,and numeric values represent positional linkages which may besubstituted with any other positional linkages as required.

In yet still another preferred embodiment, the immunogenic compositioncomprises a GPI inositolglycan domain wherein said GPI inositolglycandomain comprises the structure

EtN-P-(Manα1,2)-6Manα1, 2Mα1, 6Manα1, 4GlcNH₂α1-myo-inositol-1,2cyclic-phosphate

or derivative or equivalent thereof wherein EtN is ethanolamine, P isphosphate and M is mannose.

Even more preferably, the immunogenic composition comprises a GPIinositolglycan domain wherein said GPI inositolglycan domain comprisesthe structure

NH₂—CH₂—CH₂—PO₄-(Manα1-2) 6Manα1-2Manα1-6Manα1-4GlcNH₂-6myo-inositol-1,2 cyclic-phosphate

or derivative or equivalent thereof.

The term “mammal” includes humans, primates, livestock animals (eg.horses, cattle, sheep, pigs, donkeys), laboratory test animals (eg.mice, rats, rabbits, guinea pigs), companion animals (eg. dogs, cats)and captive wild animals (eg. kangaroos, deer, foxes). Preferably, themammal is a human or laboratory test animal. Even more preferably, themammal is a human.

The mammal undergoing treatment may be a human or animal in need oftherapeutic or prophylactic treatment for a disease condition or apotential disease condition.

Without limiting this aspect of the present invention, administration ofsaid immunogenic composition may act to result in production ofantibodies which either prevent manifestation of symptoms such astoxicity and immunosuppression or which affect the parasite directly,for example, by killing the parasite via binding to its surface andinhibiting its growth, development or the onward progression of theoverall infection.

An “effective amount” means an amount necessary at least partly toattain the desired immune response, or to prevent or to delay the onsetor inhibit progression or halt altogether, the onset or progression of aparticular condition being treated. This amount varies depending uponthe health and physical condition of the individual to be treated, thetaxonomic group of individual to be treated, the capacity of theindividual's immune system to synthesize antibodies, the degree ofprotection desired, the formulation of the vaccine, the assessment ofthe medical situation, and other relevant factors. It is expected thatthe amount will fall in a relatively broad range that can be determinedthrough routine trials.

Reference herein to “treatment” and “prophylaxis” is to be considered inits broadest context. The term “treatment” does not necessarily implythat a mammal is treated until total recovery. Similarly, “prophylaxis”does not necessarily mean that the subject will not eventually contracta disease condition. Accordingly, treatment and prophylaxis includeamelioration of the symptoms of a particular condition or preventing orotherwise reducing the risk of developing a particular condition. Theterm “prophylaxis” may be considered as reducing the severity of onsetof a particular condition. “Treatment” may also reduce the severity ofan existing condition or the frequency of acute attacks (for example,reducing the severity of initial infection).

In accordance with these methods, the modulatory agent defined inaccordance with the present invention may be coadministered with one ormore other compounds or molecules. By “coadministered” is meantsimultaneous administration in the same formulation or in two differentformulations via the same or different routes or sequentialadministration by the same or different routes. By “sequential”administration is meant a time difference of from seconds, minutes,hours or days between the administration of the two types of molecules,These molecules may be administered in any order.

In a related aspect, the present invention provides a method for thetreatment and/or prophylaxis of a mammalian disease conditioncharacterised by a microorganism infection, said method comprisingadministering to said mammal an effective amount of an immunogeniccomposition which composition comprises a molecule capable of inducingan immune response directed to the inositolglycan domain of a GPI, butsubstantially incapable of inducing an immune response directed to thelipid domain of a GPI, for a time and under conditions sufficient forsaid immune response to reduce, inhibit or otherwise alleviate any oneor more symptoms associated with said microorganism infection.

More particularly, the present invention is directed to a method for thetreatment and/or prophylaxis of a mammalian disease conditioncharacterised by a microorganism infection said method comprisingadministering to said mammal an effective amount of an immunogeniccomposition which composition comprises a modified GPI molecule orderivative or equivalent thereof and which modified GPI moleculecomprises insufficient lipidic domain to induce or elicit an immuneresponse directed to a GPI lipidic domain for a time and underconditions sufficient for said immune response to reduce, inhibit orotherwise alleviate any one or more symptoms associated with saidmicroorganism infection.

Preferably, said disease condition is malaria and said microorganism isPlasmodium falciparum.

In accordance with this preferred aspect of the present invention, theimmunogenic composition preferably comprises a GPI inositolglycan domainwherein said GPI inositolglycan domain comprises the structure

ethanolamine-phosphate-(Manα1,2)-Manα1,2Manα1,6Manα1,4GlcN-myo-inositolphosphoglycerol

or derivative or equivalent thereof.

In another preferred embodiment, the subject inositolglycan domaincomprises the structure

X₁—X₂—X₃—X₄-ethanolamine-phosphate-(Manα1,2)-Manα1,2Manα1,6Manα1,4GlcN-myo-inositolphosphoglycerol

wherein X₁, X₂, X₃ and X₄ are any 4 amino acids, or derivative orequivalent of said GPI inositolglycan domain.

In still another preferred embodiment, the subject inositolglycan domaincomprises a structure selected from:

EtN-P-[Mα2]Mα2 Mα6 Mα4Gα6Ino

EtN-P-[Mα2][G]Mα2 Mα6 Mα4Gα6Ino

EtN-P-[Mα2][X]Mα2 Mα6 Mα4Gα6Ino

EtN-P-[Mα2]-[EtN-P]Mα2 Mα6 Mα4Gα6Ino

EtN-P-Mα2 Mα6 Mα4G

Mα2 Mα6 Mα4G

EtN-P-Mα2 Mα6 M

EtN-P-[Mα2][G]Mα2 Mα6 Mα4G

EtN-P-[Mα2][X]Mα2 Mα6 Mα4G

EtN-P-[Mα2][EtN-P]Mα2 Mα6 Mα4G

Mα2 [Mα2][G]Mα2 Mα6 Mα4G

Mα2 [Mα2][X]Mα2 Mα6 Mα4G

Mα2 [Mα2][EtN-P]Mα6 Mα4G

Mα6 Mα4Gα6Ino

Mα2 Mα6 Mα4Gα6Ino

Mα2 [Mα2]Mα6 Mα4Gα6Ino

Mα2 [Mα2][G]Mα2 Mα4Gα6Ino

Mα2 [Mα2][X]Mα6 Mα4Gα6Ino

EtN-P-[Mα2][G]Mα2 Mα6 M

EtN-P-[Mα2][X]Mα2 Mα6 M

EtN-P-[Mα2][EtN-P]Mα2 Mα6 M

Mα2 [Mα2][G]Mα2 Mα6 M

Mα2 [Mα2][X]Mα2 Mα6 M

Mα2 [Mα2][EtN-P]Mα6 M

Mα6 Mα4G

EtN-P-[Mα2][G]Mα2 M

EtN-P-[Mα2][X]Mα2 M

EtN-P-[Mα2][EtN-P]Mα2 M

or derivative or equivalent thereof wherein EtN is ethanolamine, P isphosphate, M is mannose, G is non-N-acetylated glucosamine, [G] is anynon-N-acetylated hexosamine, Ino is inositol orinositol-phosphoglycerol, [X] is any other substituent, α representsα-linkages which may be substituted with β-linkages wherever required,and numeric values represent positional linkages which may besubstituted with any other positional linkages as required.

In yet another aspect the present invention relates to the use of acomposition comprising a molecule capable of inducing an immune responsedirected to a microorganism GPI inositolglycan domain but substantiallyincapable of inducing an immune response directed to a lipidic domain ofGPI in the manufacture of a medicament for the therapeutic and/orprophylactic treatment of a mammalian disease condition characterised byinfection with said microorganism.

In yet still another preferred embodiment, the immunogenic compositioncomprises a GPI inositolglycan domain wherein said GPI inositolglycandomain comprises the structure

EtN-P-(Manα1,2)-6Manα1, 2Manα1, 6Manα1, 4GlcNH₂α1-myo-inositol-1,2cyclic-phosphate

or derivative or equivalent thereof wherein EtN is ethanolamine, P isphosphate and M is mannose.

Even more preferably, the immunogenic composition comprises a GPIinositolglycan domain wherein said GPI inositolglycan domain comprisesthe structure

NH₂—CH₂—CH₂—PO₄-(Manα1-2) 6Manα1-2Manα1-6Manα1-4GlcNH₂-6myo-inositol-1,2 cyclic-phosphate

or derivative or equivalent thereof.

Accordingly, another aspect the present invention relates to the use ofan immunogenic composition comprising a Plasmodium GPI inositolglycandomain or derivative or equivalent thereof which inositolglycan domaincomprises insufficient lipidic domain of a Plasmodium GPI to elicit orinduce an immune response directed to a GPI lipidic domain in themanufacture of a medicament for the therapeutic and/or prophylactictreatment of a mammalian disease condition characterised by infectionwith said Plasmodium.

Preferably said disease condition is malaria.

The present invention should also be understood to extend to immunogeniccompositions for use in the methods as hereinbefore defined.

Accordingly, in a related aspect, the present invention is directed to acomposition capable of inducing an immune response directed to amicroorganism, said composition comprising a molecule capable ofinducing an immune response against a microorganism GPI inositolglycandomain but substantially incapable of inducing an immune response to alipidic domain of a GPI.

More particularly, the present invention is directed to a compositioncapable of inducing an immune response directed to a microorganism saidcomposition comprising a modified GPI molecule or derivative orequivalent thereof which modified GPI molecule comprises insufficientlipidic domain to induce or elicit an immune response directed to a GPIlipidic domain.

Preferably, said modified GPI molecule is the inositolglycan domainportion of GPI.

Even more preferably, said microorganism is a parasite and said parasiteis Plasmodium.

In accordance with this preferred aspect of the present invention, theimmunogenic composition preferably comprises a GPI inositolglycan domainwherein said GPI inositolglycan domain comprises the structure

ethanolamine-phosphate-(Manα1,2)-Manα1,2Manα1,6Manα1,4GlcN-myo-inositolphosphoglycerol

or derivative or equivalent thereof.

In another preferred embodiment, the subject inositolglycan domaincomprises the structure

X₁—X₂—X₃—X₄-ethanolamine-phosphate-(Manα1,2)-Manα1,2Manα1,6Manα1,4GlcN-myo-inositolphosphoglycerol

wherein X₁, X₂, X₃ and X₄ are any 4 amino acids, or derivative orequivalent of said GPI inositolglycan domain.

In still another preferred embodiment, the subject inositolglycan domaincomprises a structure selected from:

EtN-P-[Mα2]Mα2 Mα6 Mα4Gα6Ino

EtN-P-[Mα2][G]Mα2 Mα6 Mα4Gα6Ino

EtN-P-[Mα2][X]Mα2 Mα6 Mα4Gα6Ino

EtN-P-[Mα2][EtN-P]Mα2 Mα6 Mα4Gα6Ino

EtN-P-Mα2 Mα6 Mα4G

Mα2 Mα6 Mα4G

EtN-P-Mα2 Mα6 M

EtN-P-[Mα2][G]Mα2 Mα6 Mα4G

EtN-P-[Mα2][X]Mα2 Mα6 Mα4G

EtN-P-[Mα2][EtN-P]Mα2 Mα6 Mα4G

Mα2 [Mα2][G]Mα2 Mα6 Mα4G

Mα2 [Mα2][X]Mα2 Mα6 Mα4G

Mα2 [Mα2][EtN-P]Mα6 Mα4G

Mα2 Mα4Gα6Ino

Mα2 Mα6 Mα4Gα6Ino

Mα2 [Mα2]Mα6 Mα4Gα6Ino

Mα2 [Mα2][G]Mα6 Mα4Gα6Ino

Mα2 [Mα2][X]Mα2 Mα4Gα6Ino

EtN-P-[Mα2][G]Mα2 Mα6 M

EtN-P-[Mα2][X]Mα2 Mα6 M

EtN-P-[Mα2][EtN-P]Mα2 Mα6 M

Mα2 [Mα2][G]Mα2 Mα6 M

Mα2 [Mα2][X]Mα2 Mα6 M

Mα2 [Mα2][EtN-P]Mα6 M

Mα2 Mα6 M

Mα6 Mα4G

EtN-P-[Mα2][G]Mα2 M

EtN-P-[Mα2][X]Mα2 M

EtN-P-[Mα2][EtN-P]Mα2 M

or derivative or equivalent thereof wherein EtN is ethanolamine, P isphosphate, M is mannose, G is non-N-acetylated glucosamine, [G] is anynon-N-acetylated hexosamine, Ino is inositol orinositol-phosphoglycerol, [X] is any other substituent, α representsα-linkages which may be substituted with β-linkages wherever required,and numeric values represent positional linkages which may besubstituted with any other positional linkages as required.

In yet still another preferred embodiment, the immunogenic compositioncomprises a GPI inositolglycan domain wherein said GPI inositolglycandomain comprises the structure

EtN-P-(Manα1,2)-6Mα1,2Manα1, 6Manα1, 4GlcNH₂α1-myo-inositol-1,2cyclic-phosphate

or derivative or equivalent thereof wherein EtN is ethanolamine, P isphosphate and M is mannose.

Even more preferably, the immunogenic composition comprises a GPIinositolglycan domain wherein said GPI inositolglycan domain comprisesthe structure

NH₂—CH₂—CH₂—PO₄-(Manα1-2) 6Manα1-2Manα1-6Manα1-4GlcNH₂-6myo-inositol-1,2 cyclic-phosphate

or derivative or equivalent thereof.

Yet another aspect of the present invention relates to a vaccinecomposition comprising as the active component a molecule capable ofinducing an immune response directed to a microorganism GPIinositolglycan domain but substantially incapable of inducing an immuneresponse directed to a lipidic domain of a GPI, as broadly describedabove, together with one or more pharmaceutically acceptable carriersand/or diluents.

More particularly, the present invention relates to a vaccinecomposition comprising as the active component a modified GPI moleculeor derivative or equivalent thereof which modified GPI molecule orderivative or equivalent thereof which modified GPI molecule comprisesinsufficient lipidic domain to induce or elicit an immune responsedirected to a GPI lipidic domain.

Preferably said modified GPI molecule is a GPI inositoglycan domain.

More preferably, said GPI inositolglycan domain is a parasite GPIinositolglycan domain and even more preferably a Plasmodium GPIinositolglycan domain.

Most preferably, said Plasmodium is P. falciparum.

In a most preferred embodiment, said molecule is a GPI inositolglycandomain comprising the structure

ethanolamine-phosphate-(Manα1,2)-Manα1,2Manα1,6Manα1,4GlcN-phosphatidyl-myo-inositolphosphoglycerol.

In another most preferred embodiment said molecule is a GPIinositolglycan domain comprising the structure

X₁—X₂—X₃—X₄-ethanolamine-phosphate-(Manα1,2)-Manα1,2Manα1,6Manα1,4GlcN-myo-inositolphosphoglycerol

wherein X₁, X₂, X₃, X₄, are any 4 amino acids.

In still another preferred embodiment, the subject inositolglycan domaincomprises a structure selected from:

EtN-P-[Mα2]Mα2 Mα6 Mα4Gα6Ino

EtN-P-[Mα2][G]Mα2 Mα6 Mα4Gα6Ino

EtN-P-[Mα2][X]Mα2 Mα6 Mα4Gα6Ino

EtN-P-[Mα2][EtN-P]Mα2 Mα6 Mα4Gα6Ino

EtN-P-Mα2 Mα6 Mα4G

Mα2 Mα6 Mα4G

EtN-P-Mα2 Mα6 M

EtN-P-[Mα2][G]Mα2 Mα6 Mα4G

EtN-P-[Mα2][X]Mα2 Mα6 Mα4G

EtN-P-[Mα2][EtN-P]Mα2 Mα6 Mα4G

Mα2 [Mα2][G]Mα2 Mα6 Mα4G

Mα2 [Mα2][X]Mα2 Mα6 Mα4G

Mα2 [Mα2][EtN-P]Mα6 Mα4G

Mα6 Mα4Gα6Ino

Mα2 Mα6 Mα4Gα6Ino

Mα2 [Mα2]Mα6 Mα4Gα6Ino

Mα2 [Mα2][G]Mα6 Mα4Gα6Ino

Mα2 [Mα2][X]Mα6 Mα4Gα6Ino

EtN-P-[Mα2][G]Mα2 Mα6 M

EtN-P-[Mα2][X]Mα2 Mα6 M

EtN-P-[Mα2][EtN-P]Mα2 Mα6 M

Mα2 [Mα2][G]Mα2 Mα6 M

Mα2 [Mα2][X]Mα2 Mα6 M

Mα2 [Mα2][EtN-P]Mα6 M

Mα6 Mα4G

EtN-P-[Mα2][G]Mα2 M

EtN-P-[Mα2][X]Mα2 M

EtN-P-[Mα2][EtN-P]Mα2 M

or derivative or equivalent thereof wherein EtN is ethanolamine, P isphosphate, M is mannose, G is non-N-acetylated glucosamine, [G] is anynon-N-acetylated hexosamine, Ino is inositol orinositol-phosphoglycerol, [X] is any other substituent, α representsα-linkages which may be substituted with β-linkages wherever required,and numeric values represent positional linkages which may besubstituted with any other positional linkages as required.

In yet still another preferred embodiment, the immunogenic compositioncomprises a GPI inositolglycan domain wherein said GPI inositolglycandomain comprises the structure

EtN-P-(Manα1,2)-6Mα1, 2Manα1, 6Manα1, 4GlcNH₂α1-myo-inositol-1,2cyclic-phosphate

or derivative or equivalent thereof wherein EtN is ethanolamine, P isphosphate and M is mannose.

Even more preferably, the immunogenic composition comprises a GPIinositolglycan domain wherein said GPI inositolglycan domain comprisesthe structure

NH₂—CH₂—CH₂—PO₄-(Manα1-2) 6Manα1-2Manα1-6Manα1-4GlcNH₂-6myo-inositol-1,2 cyclic-phosphate

or derivative or equivalent thereof.

Still another aspect of the present invention is directed to apharmaceutical composition comprising a molecule capable of inducing animmune response directed to a microorganism GPI inositolglycan domainbut substantially incapable of inducing an immune response directed to alipidic domain of a GPI, as broadly described above, together with oneor more pharmaceutically acceptable carriers and/or diluents.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) and sterile powders for theextemporaneous preparation of sterile injectable solutions or dispersionor may be in the form of a cream or other form suitable for topicalapplication. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol and liquid polyethylene glycol,and the like), suitable mixtures thereof, and vegetable oils. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of superfactants. The preventions of theaction of microorganisms can be brought about by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredient into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

When the active ingredients are suitably protected they may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or it may be enclosed in hard or soft shell gelatincapsule, or it may be compressed into tablets, or it may be incorporateddirectly with the food of the diet. For oral therapeutic administration,the active compound may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 1% by weight of active compound.The percentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 5 to about 80% of theweight of the unit. The amount of active compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained. Preferred compositions or preparations according to thepresent invention are prepared so that an oral dosage unit form containsbetween about 0.1 μg and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain thecomponents as listed hereafter: a binder such as gum, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, lactose or saccharin may be added or a flavouringagent such as peppermint, oil of wintergreen, or cherry flavouring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavouring such as cherry or orange flavour. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound(s) may be incorporated intosustained-release preparations and formulations.

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, use thereof in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the novel dosageunit forms of the invention are dictated by and directly dependent on(a) the unique characteristics of the active material and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active material for the treatment ofdisease in living subjects having a diseased condition in which bodilyhealth is impaired as herein disclosed in detail.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form as hereinbeforedisclosed. A unit dosage form can, for example, contain the principalactive compound in amounts ranging from 0.5 μg to about 2000 mg.Expressed in proportions, the active compound is generally present infrom about 0.5 μg to about 2000 mg/ml of carrier. In the case ofcompositions containing supplementary active ingredients, the dosagesare determined by reference to the usual dose and manner ofadministration of the said ingredients.

The pharmaceutical composition may also comprise genetic molecules suchas a vector capable of transfecting target cells where the vectorcarries a nucleic acid molecule capable of expressing, for example, afunctional equivalent to a GPI inositolglycan domain or derivativethereof. The vector may, for example, be a viral vector and it may beadministered by any suitable method including, for example transfectiondirectly into the cells of the mammal being treated or transfection intoa host cell, such as a bacterium, yeast or attenuated parasite, which isthen introduced into the mammal.

Administration of the immunogenic GPI inositolglycan domain of thepresent invention induces antibody production and in particular IgGproduction. Said antibodies are involved in inhibiting, halting ordelaying the onset or progression of symptoms associated withmicroorganism infection such as, for example, pathological responses toa parasitic infection. Said antibodies function, for example, byneutralizing parasite induced TNF induction or by direct antiparasiticeffect such as killing the parasite by binding to its surface andinhibiting its growth or development or otherwise inhibiting its onwardprogression. Antibodies directed to the GPI inositolglycan domain orderivatives thereof may therefore also be utilized in treating parasiticinfections therapeutically or prophylactically.

Accordingly, another aspect of the present invention is directed toantibodies to GPI inositolglycan domains but substantially incapable ofinteracting with the lipidic domain of a GPI.

Such antibodies may be monoclonal or polyclonal, may be of any isotyopeand may be selected from naturally occurring antibodies to endogenous orexogenous GPI inositolglycan domains or may be specifically raised toGPI inositolglycan domains. Antibodies may also have been raised againstantigens other than the GPI inositolglycan domain but are cross-reactivewith one or more epitopes of the GPI inositolglycan domain. In the caseof antibodies raised to the GPI inositolglycan domain, a GPIinositolglycan may first need to be associated with a carrier moleculeas hereinbefore described.

The antibodies and/or GPI inositolglycan domains of the presentinvention are particularly useful as therapeutic or diagnostic agents.For example, a GPI inositolglycan domain can be used to screen fornaturally occurring antibodies to GPI inositolglycan domain. These mayoccur, for example in some infectious and autoimmune diseases.Alternatively, specific antibodies can be used to screen for GPIinositolglycan domains. Techniques for such assays are well known in theart and include, for example, sandwich assays, ELISA, Western blot andflow cytometry. Knowledge of GPI inositolglycan domain levels may beimportant for diagnosis of certain diseases, such as parasiticinfections, autoimmune diseases (e.g. Type 1 diabetes), degenerativediseases (e.g. Type 2 diabetes) and somatically acquired genetic defects(e.g. Paroxysmal Nocturnal Haemoglobinurea) or for monitoring certaintherapeutic protocols. Said antibodies would be useful as research toolsor reagents for the detection of GPI inositolglycan domains. Saidantibodies would also be important for example as a means for screeningfor levels of GPI inositolglycan domains in cell extract or otherbiological fluid or purifying a GPI made by recombinant means fromculture supernatant fluids. Techniques for the assays contemplatedherein and known in the art and include, for example, sandwich assaysand ELISA, Western blot and affinity chromatography.

Antibodies to GPI inositolglycan domain of the present invention may bemonoclonal or polyclonal. Alternatively, fragments of antibodies may beused such as Fab fragments. Furthermore, the present invention extendsto recombinant and synthetic antibody, to antibody hybrid and tohumanised antibody. A “synthetic antibody” is considered herein toinclude fragments and hybrids of antibodies. The antibodies of thisaspect of the present invention are particularly useful forimmunotherapy and immunoprophylaxis and may also be used as a diagnostictool for assessing, for example, parasitic infection or for monitoringthe program of therapeutic regimen.

It is within the scope of this invention to include any secondantibodies (monoclonal, polyclonal or fragments of antibodies orsynthetic antibodies) directed to the first mentioned antibodiesdiscussed above. Both the first and second antibodies may be used indetection assays or a first antibody may be used with a commerciallyavailable anti-immunoglobulin antibody. An antibody as contemplatedherein includes any antibody specific to any region of the GPIinositolglycan domain.

Both polyclonal and monoclonal antibodies are obtainable by immunizationwith the GPI inositolglycan domain and are utilizable for immunoassays.The methods of obtaining both types of sera are well known in the art.Polyclonal sera are less preferred but are relatively easily prepared byinjection of a suitable laboratory animal with an effective amount of aGPI inositolglycan domain, or antigenic parts thereof, collecting serumfrom the animal, and isolating specific sera by any of the knownimmunoadsorbent techniques. Although antibodies produced by this methodare utilizable in virtually any type of immunoassay, they are generallyless favoured because of the potential heterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is particularlypreferred because of the ability to produce them in large quantities andthe homogeneity of the product. The preparation of hybridoma cell linesfor monoclonal antibody production derived by fusing an immortal cellline and lymphocytes sensitized against the immunogenic preparation canbe done by techniques which are well known to those who are skilled inthe art.

Yet another aspect of the present invention relates to a pharmaceuticalcomposition comprising an antibody directed to a GPI inositolglycandomain together with one or more pharmaceutically acceptable carriers ordiluents as hereinbefore described.

A further aspect of the present invention relates to the use of theantibodies of the present invention in relation to disease conditions.For example, the present invention is particularly useful but in no waylimited to use in treating parasitic infections, their symptoms andpathologies.

Accordingly, another aspect of the present invention relates to a methodof inhibiting, halting or delaying the onset of progression of amammalian disease condition characterised by a microorganism infectionsaid method comprising administering to said mammal an effective amountof an antibody has hereinbefore described.

Preferably said disease condition is a parasite infection and mostpreferably malaria.

In yet another aspect the present invention relates to the use of anantibody in the manufacture of a medicament for inhibiting, halting ordelaying the onset or progression of a disease condition characterisedby the infection of a mammal by a microorganism.

Preferably said disease condition is a parasite infection and mostpreferably malaria.

In yet another further aspect, the present invention envisagesdiagnostic, monitoring, screening or other qualitative or quantitativeantigen based assessments of either an immune response or a populationof immunointeractive molecules directed to a microorganism, such as aparasite, utilizing the GPI inositolglycan molecules hereinbeforedisclosed, in particular the synthetic GPI inositolglycan moleculedisclosed herein.

Accordingly, another aspect of the present invention provides a methodfor detecting, in a biological sample, an immunointeractive moleculedirected to a microorganism said method comprising contacting saidbiological sample with a molecule comprising said microorganism GPIinositolglycan domain or a derivative or equivalent thereof andqualitatively and/or quantitatively screening for said GPIinositolglycan domain-immunointeractive molecule complex formation.

In a related aspect, the present invention provides a method fordetecting, monitoring or otherwise assessing an immune response directedto a microorganism in a subject said method comprising contacting abiological sample, from said subject, with a molecule comprising saidmicroorganism GPI inositolglycan domain or a derivative or equivalentthereof and qualitatively and/or quantitatively screening for GPIinositolglycan domain-immunointeractive molecule complex formation.

Reference to “GPI inositolglycan domain” should be understood to havethe same meaning as hereinbefore provided.

More particularly, according to these aspects of the present inventionsaid GPI inositolglycan domain substantially does not contain a portioncapable of inducing an immune response directed to a lipidic domain ofsaid GPI. Most preferably, said GPI molecule is the inositolglycandomain portion of GPI or derivative or equivalent thereof.

Reference to “derivatives” and “equivalents” should be understood tohave the same meaning as hereinbefore provided.

The term “microorganism” should be understood in its broadest sense andincludes, for example, the parasitic and fungal taxa Plasmodium,Trypanosoma, Leishmania, Toxoplasma and Canidida. “Microorganism” shouldalso be understood to extend to molecules which are secreted by or shedfrom the subject organism. This would include for example, toxinmolecules or molecules which are cleared from the surface of themicroorganism. Preferably, the GPI inositolglycan domain suitable foruse in the present invention is a parasite GPI inositolglycan domain andeven more preferably a Plasmodium GPI inositolglycan domain.

In one aspect, the present invention therefore more preferably providesa method for detecting, in a biological sample, an immunointeractivemolecule directed to Plasmodium said method comprising contacting saidbiological sample with the inositolglycan domain portion of a PlasmodiumGPI or derivative or equivalent thereof and qualitatively and/orquantitatively screening for GPI inositolglycan domain-immunointeractivemolecule complex formation.

In a related aspect, the present invention more preferably provides amethod for detecting, monitoring or otherwise assessing an immuneresponse directed to Plasmodium in a subject said method comprisingcontacting a biological sample, from said subject, with theinositolglycan domain portion of a Plasmodium GPI or derivative orequivalent thereof and qualitatively and/or quantitatively screening forGPI inositolglycan domain-immunointeractive molecule complex formation.

Even more preferably, said Plasmodium is P. falciparum.

In one embodiment of these preferred aspects, said GPI inositolglycandomain comprises the structure

ethanolamine-phosphate-(Manα1,2)-Manα1,2Manα1,6Manα1,4GlcN-myo-inositolphosphoglycerol

or derivative or equivalent thereof.

In another embodiment of these preferred aspects said GPI inositolglycandomain comprises the structure

X₁—X₂—X₃—X₄-ethanolamine-phosphate-(Manα1,2)-Manα1,2Manα1,6Manα1,4GlcN-myo-inositolphosphoglycerol

wherein X₁, X₂, X₃ and X₄ are any 4 amino acids, or derivative orequivalent of said GPI inositolglycan domain.

In still another embodiment of these preferred aspects said GPIinositolglycan domain comprises the structure

EtN-P-[Mα2]Mα2 Mα6 Mα4Gα6Ino

EtN-P-[Mα2][G]Mα2 Mα6 Mα4Gα6Ino

EtN-P-[Mα2][X]Mα2 Mα6 Mα4Gα6Ino

EtN-P-[Mα2][EtN-P]Mα2 Mα6 Mα4Gα6Ino

EtN-P-Mα2 Mα6 Mα4G

Mα2 Mα6 Mα4G

EtN-P-Mα2 Mα6 M

EtN-P-[Mα2][G]Mα2 Mα6 Mα4G

EtN-P-[Mα2][X]Mα2 Mα6 Mα4G

EtN-P-[Mα2][EtN-P]Mα2 Mα6 Mα4G

Mα2 [Mα2][G]Mα2 Mα6 Mα4G

Mα2 [Mα2][X]Mα2 Mα6 Mα4G

Mα2 [Mα2][EtN-P]Mα6 Mα4G

Mα6 Mα4Gα6Ino

Mα2 Mα6 Mα4Gα6Ino

Mα2 [Mα2]Mα6 Mα4Gα6Ino

Mα2 [Mα2][G]Mα6 Mα4Gα6Ino

Mα2 [Mα2][X]Mα6 Mα4Gα6Ino

EtN-P-[Mα2][G]Mα2 Mα6 M

EtN-P-[Mα2][X]Mα2 Mα6 M

EtN-P-[Mα2][EtN-P]Mα2 Mα6 M

Mα2 [Mα2][G]Mα2 Mα6 M

Mα2 [Mα2][X]Mα2 Mα6 M

Mα2 [Mα2][EtN-P]Mα6 M

Mα2 Mα6 M

Mα6 Mα4G

EtN-P-[Mα2][G]Mα2 M

EtN-P-[Mα2][X]Mα2 M

EtN-P-[Mα2][EtN-P]Mα2 M

or derivative or equivalent thereof wherein EtN is ethanolamine, P isphosphate, M is mannose, G is non-N-acetylated glucosamine, [G] is anynon-N-acetylated hexosamine, Ino is inositol orinositol-phosphoglycerol, [X] is any other substituent, α representsα-linkages which may be substituted with β-linkages wherever required,and numeric values represent positional linkages which may besubstituted with any other positional linkages as required.

Any of these structures may be further modified by substituents ofpositive, negative or neutral charge such as phosphates,phosphoglycerol, hexosamines, amino acids, thiols etc in any positionand with any type of linkage. These structures may be further modifiedby addition of any number of amino acids for the purpose of providing alinkage sequence.

Most preferably, said GPI inositolglycan domain is a synthetic moleculeand comprises the structure

EtN-P-(Manα1,2)-6Manα1, 2Manα1, 6Manα1, 4GlcNH₂α1-myo-inositol-1,2cyclic-phosphate

or derivative or equivalent thereof wherein EtN is ethanolamine, P isphosphate and M is mannose; or

NH₂—CH₂—CH₂—PO₄-(Manα1-2) 6Manα1-2Manα1-6Manα1-4GlcNH₂-6myo-inositol-1,2 cyclic-phosphate

or derivative or equivalent thereof.

Reference to “biological sample” should be understood as a reference toany sample of biological material derived from an individual such, butnot limited to, mucus, stool, urine, blood, serum, cell extract, biopsyspecimens and fluid which has been introduced into the body of anindividual and subsequently removed such as, for example, the salinesolution extracted from the lung following lung lavage or the solutionretrieved from an enema wash. The biological sample which is testedaccording to the method of the present invention may be tested directlyor may require some form of treatment prior to testing. For example, abiopsy sample may require homogenisation or sectioning prior to testing.Alternatively, the sample may require some other form of treatment inorder to render it suitable for analysis. Preferably, the subject sampleis a blood sample.

The “immunointeractive molecule” is any molecule having specificity andbinding affinity for the GPI inositolglycan domain or its antigenicparts. Although the preferred immunointeractive molecule is animmunglobulin molecule, the present invention extends to otherimmunointeractive molecules such as antibody fragments, single chainantibodies, deimmunized including humanized antibodies and T-cellassociated antigen-binding molecules (TABMs). Most preferably, theimmunointeractive molecule is an antibody such as a polyclonal ormonoclonal antibody. It should be understood that the subjectimmunointeractive molecule may be linked, bound or otherwise associatedto any other proteinaceous or non-proteinaceous molecule or cell. Mostpreferably, the antibody is a monoclonal antibody.

The immunointeractive molecule exhibits specificity for GPI or moreparticularly an antigenic determinant or epitope of the GPIinositolglycan domain. An antigenic determinant or epitope of the GPIinositolglycan domain includes that part of the molecule to which animmune response can be directed. The antigenic determinant or epitope ispreferably a B-cell epitope but may be, where appropriate, a T-cellreceptor binding peptide. The term “antigenic part” includes anantigenic determinant or epitope.

One aspect of the present invention therefore most preferably provides amethod for detecting, in a biological sample, an antibody directed toPlasmodium said method comprising contacting said biological sample withthe inositolglycan domain portion of a Plasmodium GPI or a derivative orequivalent thereof and qualitatively and/or quantitatively screening forGPI inositolglycan domain-antibody complex formation.

A related aspect of the present invention most preferably provides amethod for detecting, monitoring, or otherwise assessing an immuneresponse directed to Plasmodium in a subject said method comprisingcontacting a biological sample, from said subject, with theinositolglycan domain portion of a Plasmodium GPI or a derivative orequivalent thereof and qualitatively and/or quantitatively screening forGPI inositolglycan domain-antibody complex formation.

Most preferably, said Plasmodium is P. falcimparum.

Reference to “immune response” should be understood as a reference toany form of specific immune response. Preferably, the subject responseis a B cell/antibody response. It should also be understood that thesubject immune response may be at any stage of development. For example,in a most preferred embodiment, one screens for secreted anti-Plasmodiumantibodies (i.e. the onset of the effector B cell response). However,one may also apply the method at a cellular level to screen for orotherwise analyse the expansion and/or maturity of the B cellsub-population which is directed to the Plasmodium GPI inositolglycandomain and will therefore provide protective immunity. Although thepreferred method is to assess the onset of an immune response in asubject it should be understood that the present invention alsoencompasses the analysis of in vitro-based immune responses, such asthose which occur in the context of generating or analysing B cellhybridomas.

Reference to “detecting, monitoring or otherwise assessing” an immuneresponse or immunointeractive molecule should be understood as areference to any form of analysis which one may seek to perform in thecontext of the subject immune response including, but not limited to:

-   (i) assessing the existence, onset or degree of clinical immunity of    individuals. This extends to assessing the seroconversion of    vaccinated individuals as well as determining the degree of    naturally acquired anti-toxin antibodies. As shown in Example 18,    the GPI glycan can be used to detect antibodies from glycan    immunized mice. Identical glycan:protein coupling methods have also    been used to generate glycan:protein constructs capable of detecting    IgG in the sera of humans exposed to malaria. These antibodies    confer protective clinical immunity to malaria and individuals may    acquire such immunity after exposure to malaria infection. The    detection of such antibodies may be useful to indicate the clinical    immune status of individuals. For example, following mass    vaccination campaigns it is desirable to assess the seroconversion    of target groups and the GPI glycan will have utility as an    indicator of such seroconversion.-   (ii) determining whether an immune response is of a primary (e.g.    IgM) or secondary (IgG) type. This can have particular relevance to    postulating whether an individual's parasitic infection status is    acute or chronic.-   (iii) monitoring disease progression, for example in the context of    assessing the effectiveness of a therapeutic response-   (iv) detecting, and thereby potentially facilitating the isolation    and analysis of, antibodies which neutralise parasite derived    toxins, such as the malaria toxin.-   (v) detecting, and thereby potentially facilitating the isolation    and analysis of, monoclonal or polyclonal antibodies of animal or    human origin which are directed towards the parasite GPI    inositolglycan domain and which would therefore exhibit utility in    the treating of disease.

The present invention is therefore useful as a one off test or as anongoing monitor in either in vitro or in vivo contexts. Accordingly, themethod of the present invention should be understood to extend tomonitoring for increases or decreases in levels of GPI antibody complexformation relative either to normal levels (as hereinafter defined) orrelative to one or more earlier levels as determined for a givensituation. In this regard, “qualitatively and/or quantitativelyscreening for” GPI-antibody complex formation should be understood toencompass both screening for the presence or absence of complexformation or screening for the level of complex formation either for thepurpose of obtaining an absolute quantitative value or for the purposeof correlation with normal levels or earlier obtained levels. The“normal” level is the level of complex in a biological samplecorresponding to an individual who either is not infected or has not yetdeveloped an immune response. This “normal” level may be a standardresult which reflects individual or collective results obtained fromhealthy individuals, other than the patient in issue. Said “normallevel” may be a discrete level or a range of levels. Individualsexhibiting complex levels higher than the normal range are generallyregarded as having undergone the onset or expansion of an immuneresponse directed to a parasite infection.

The method of the present invention has widespread applications asdetailed hereinbefore. Also detailed hereinbefore is the fact that thescreening methodology may be performed either qualitatively orquantitatively. Although it is likely that quantitative analyses will bepreferred since it provides information in relation to the occurrenceof, or not, of an immune response or the presence of a particularpopulation of immunointeractive molecules, qualitative analyses may alsobe of value. In particular, since antibodies to parasite derived cellmolecules are not normally found in the blood of uninfected individuals,a test directed to assessing the presence or not of the subjectimmunointeractive molecules will provide useful information. It willalso provide scope for establishing extremely simple and inexpensivescreening procedures.

Methods of designing and performing such diagnostic screening assayswould be well known to the person of suitable skill in the art andinclude, but are not limited to:

-   (i) In vivo detection of complex formation. Molecular Imaging may be    used following administration of imaging reagents capable of    disclosing altered levels of immunointeractive molecule expression    product in the subject.-    Molecular imaging (Moore, A., Basilion, J., Chiocca, E., and    Weissleder, R., BBA, 1402:239-249, 1988; Weissleder, R., Moore, A.,    Ph.D., Mahmood-Bhorade, U., Benveniste, H., Chiocca, E. A.,    Basilion, J. P. Nature Medicine, 6:351-355, 2000) is the in vivo    imaging of molecular expression that correlates with the    macro-features currently visualized using “classical” diagnostic    imaging techniques such as X-Ray, computed tomography (CT), MRI or    Positron Emission Tomography (PET).-   (ii) Measurement of altered complex levels in a suitable biological    sample, either qualitatively or quantitatively, for example by    immunoassay. For example, a secondary antibody having a reporter    molecule associated therewith, may be utilised in immunoassays to    detect complex formation. Such immunoassays include but are not    limited to radioimmunoassays (RIAs), enzyme-linked immunosorbent    assays (ELISAs) and immunochromatographic techniques (ICTs), Western    blotting which are well known to those of skill in the art. For    example, reference may be made to “Current Protocols in Immunology”,    1994 which discloses a variety of immunoassays which may be used in    accordance with the present invention. Immunoassays may include    competitive assays. It will be understood that the present invention    encompasses qualitative and quantitative immunoassays.-    Suitable immunoassay techniques are described, for example, in U.S.    Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. These include both    single-site and two-site assays of the non-competitive types, as    well as the traditional competitive binding assays.-    Two-site assays are particularly favoured for use in the present    invention. A number of variations of these assays exist, all of    which are intended to be encompassed by the present invention.    Briefly, in a typical forward assay, an unlabelled antigen molecule    (i.e. GPI) is immobilized on a solid substrate and the sample to be    tested brought into contact with the bound molecule. After a    suitable period of incubation, for a period of time sufficient to    allow formation of an antibody-antigen complex, another    antigen-binding molecule, suitably a second antibody specific to the    antigen, labelled with a reporter molecule capable of producing a    detectable signal is then added and incubated, allowing time    sufficient for the formation of another complex of    antibody-antigen-labelled antibody. Any unreacted material is washed    away and the presence of the antigen is determined by observation of    a signal produced by the reporter molecule. The results may be    either qualitative, by simple observation of the visible signal, or    may be quantitated by comparing with a control sample containing    known amounts of primary antibody. Variations on the forward assay    include a simultaneous assay, in which both sample and labelled    antibody are added simultaneously to the bound antigen. These    techniques are well known to those skilled in the art, including    minor variations as will be readily apparent.-    In the typical forward assay, the GPI molecule having specificity    for the immune response effector immunointeractive molecule is    either covalently or passively bound to a solid surface. The solid    surface is typically glass or a polymer, the most commonly used    polymers being cellulose, polyacrylamide, nylon, polystyrene,    polyvinyl chloride or polypropylene. The solid supports may be in    the form of tubes, beads, discs of microplates, or any other surface    suitable for conducting an immunoassay. The binding processes are    well known in the art and generally consist of cross-linking    covalently binding or physically adsorbing, the polymer-antibody    complex is washed in preparation for the test sample. An aliquot of    the sample to be tested is then added to the solid phase complex and    incubated for a period of time sufficient and under suitable    conditions to allow binding of any antibody present to the GPI    antibody. Following the incubation period, the antigen-antibody    complex is washed and dried and incubated with a second antibody    specific for a portion of the antibody. The second antibody has    generally a reporter molecule associated therewith that is used to    indicate the binding of the second antibody to the first antibody.    The amount of labelled antibody that binds, as determined by the    associated reporter molecule, is proportional to the amount of first    antibody bound to the immobilized antigen.-    From the foregoing, it will be appreciated that the reporter    molecule associated with the antigen-binding molecule may include    the following:—    -   (a) direct attachment of the reporter molecule to the antibody;    -   (b) indirect attachment of the reporter molecule to the        antibody; i.e., attachment of the reporter molecule to another        assay reagent which subsequently binds to the antibody; and    -   (c) attachment to a subsequent reaction product of the antibody.-    The reporter molecule may be selected from a group including a    chromogen, a catalyst, an enzyme, a fluorochrome, a chemiluminescent    molecule, a paramagnetic ion, a lanthanide ion such as Europium    (Eu³⁴), a radioisotope including other nuclear tags and a direct    visual label.-    In the case of a direct visual label, use may be made of a    colloidal metallic or nonmetallic particle, a dye particle, an    enzyme or a substrate, an organic polymer, a latex particle, a    liposome, or other vesicle containing a signal producing substance    and the like.-    A large number of enzymes suitable for use as reporter molecules is    disclosed in U.S. Pat. No. 4,366,241, U.S. Pat. No. 4,843,000, and    U.S. Pat. No. 4,849,338. Suitable enzymes useful in the present    invention include alkaline phosphatase, horseradish peroxidase,    luciferase, β-galactosidase, glucose oxidase, lysozyme, malate    dehydrogenase and the like. The enzymes may be used alone or in    combination with a second enzyme that is in solution.-    Suitable fluorochromes include, but are not limited to, fluorescein    isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC),    R-Phycoerythrin (RPE), and Texas Red. Other exemplary fluorochromes    include those discussed by Dower et al., International Publication    No. WO 93/06121. Reference also may be made to the fluorochromes    described in U.S. Pat. No. 5,573,909 (Singer et al), U.S. Pat. No.    5,326,692 (Brinkley et al). Alternatively, reference may be made to    the fluorochromes described in U.S. Pat. Nos. 5,227,487, 5,274,113,    5,405,975, 5,433,896, 5,442,045, 5,451,663, 5,453,517, 5,459,276,    5,516,864, 5,648,270 and 5,723,218.-    In the case of an enzyme immunoassay, an enzyme is conjugated to    the second antibody, generally by means of glutaraldehyde or    periodate. As will be readily recognized, however, a wide variety of    different conjugation techniques exist which are readily available    to the skilled artisan. The substrates to be used with the specific    enzymes are generally chosen for the production of, upon hydrolysis    by the corresponding enzyme, a detectable colour change. Examples of    suitable enzymes include those described supra. It is also possible    to employ fluorogenic substrates, which yield a fluorescent product    rather than the chromogenic substrates noted above. In all cases,    the enzyme-labelled antibody is added to the first antibody-antigen    complex, allowed to bind, and then the excess reagent washed away. A    solution containing the appropriate substrate is then added to the    complex of antibody-antigen-antibody. The substrate will react with    the enzyme linked to the second antibody, giving a qualitative    visual signal, which may be further quantitated, usually    spectrophotometrically, to give an indication of the amount of    antigen which was present in the sample.-    Alternately, fluorescent compounds, such as fluorescein, rhodamine    and the lanthanide, europium (EU), may be chemically coupled to    antibodies without altering their binding capacity. When activated    by illumination with light of a particular wavelength, the    fluorochrome-labelled antibody adsorbs the light energy, inducing a    state to excitability in the molecule, followed by emission of the    light at a characteristic colour visually detectable with a light    microscope. The fluorescent-labelled antibody is allowed to bind to    the first antibody-antigen complex. After washing off the unbound    reagent, the remaining tertiary complex is then exposed to light of    an appropriate wavelength. The fluorescence observed indicates the    presence of the antigen of interest. Immunofluorometric assays    (IFMA) are well established in the art and are particularly useful    for the present method. However, other reporter molecules, such as    radioisotope, chemiluminescent or bioluminescent molecules may also    be employed.-   (iii) Determining altered antibody production on any suitable    functional test, enzymatic test or immunological test in addition to    those detailed in point (iii)-above.

As detailed above, any suitable technique may be utilised to detect theanti-parasite antibody. The nature of the technique which is selectedfor use will largely determine the type of biological sample which isrequired for analysis. Such determinations are well within the scope ofthe person of skill in the art. A typical sample which one may seek toanalyse is a blood sample.

The present invention further provides a kit for detecting an immuneresponse to a parasitic infection. The kit may be in any convenient formbut generally comprises a solid support such as described herein adaptedto receive or comprise the GPI molecule hereinbefore defined. The kitmay also comprise reagents, reporter molecules capable of providingdetectable signals and optionally instructions for use. The kit may bein modular form wherein individual components may be separatelypurchased.

Accordingly, another aspect of the present invention is directed to amodular kit comprising one or more members wherein at least one memberis a solid support comprising a GPI molecule as hereinbefore defined.

In an alternative embodiment, the solid support comprises an array ofbinding partners for said GPI molecule.

The kit may optionally be adapted to be analyzed electronically orspectrophotometrically or fluorometrically and may further be adaptedfor high throughput screening.

In another related aspect, the determination that the GPI inositolglycandomain, in particular the synthetic form of this molecule, facilitatesthe highly valuable analysis of the onset and monitoring of the crucialaspects of the parasite specific effector immune response still furtherfacilitates rationalizing the design of anti-toxic vaccines. Human IgGdirected towards the GPI glycan exhibits epitopic specificity forsmaller partial structures within the glycan. Mapping these epitopesusing synthetic GPIs enables reduction of the number of residuesrequired for incorporation into a final vaccine. Furthermore to theextent that some epitopes are “self” and others “non-self”, the use ofGPI glycan and fragments thereof to map these specificities enhancesimmunogenicity and safety of the final product. Accordingly, thedetection of human IgG by the glycan is crucial for downstream“medicinal chemistry” rationalization of vaccine structure.

Preferably said immunointeractive molecule is an antibody.

Accordingly, the present invention should also be understood to extendto a method for analysing, designing and/or modifying an agent capableof interacting with an anti-GPI glycan immunointeractive moleculebinding site, which immunointeractive molecule is identifiable utilisingthe diagnostic methodology hereinbefore disclosed, said methodcomprising contacting said immunointeractive molecule or derivativethereof with a putative agent and assessing the degree of interactivecomplementarity of said agent with said binding site.

It should be understood that the immunointeractive molecule, for examplean antibody, which is contacted with the putative agent, for example asynthetic GPI glycan molecule, for evaluation of interactivecomplementarity may be recombinantly produced subsequently to itsidentification. However, it should also be understood that the subjectmolecule may take the form of an image based on the binding sitestructure which has been elucidated, such as an electron density map,molecular models (including, but not limited to, stick, ball and stick,space filling or surface representation models) or other digital ornon-digital surface representation models or image, which facilitatesthe analysis of molecule site: agent interactions utilising techniquesand software which would be known to those of skill in the art. Forexample, interaction analyses can be performed utilising techniques suchas Biacore real-time analysis of on and off-rates and dissociationconstants for binding of ligands (Gardsvoll, H., Dano, K., and Ploug,M., (1999), J Biol Chem, 274(53):37995-38003; Hoyer-Hansen, G.,Behrendt, N., Ploug, M., Dano, K., and Preissner, K. T., (1997), FEBSLett, 420(1):79-85; Ploug, M., (1998), Biochemistry, 37(47):16494-16505;Ploug, M., Ostergaard, S., Hansen, L. B., Holm, A., and Dano, K.,(1998), Biochemistry, 37(11):3612-3622; Ploug, M., Rahbek-Nielsen, H.,Ellis, V., Roepstorff, P., and Dano, K., (1995), Biochemistry,34(39):12524-12534; Ploug, M., Ellis, V., and Dano, K., (1994),Biochemistry, 33(30):8991-8997) and NMR perturbation studies (Stephens,R. W., Boklman, A. M., Myohanen, H. T., Reisberg, T., Tapiovaara, H.,Pedersen, N., Grondahl-Hansen, J., Llinas, M., and Vaheri, A., (1992),Biochemistry, 31:7572-7579).

Reference to “assessing the degree of interactive complementarity” of anagent with the subject molecule should be understood as a reference toelucidating any feature of interest including, but not limited to, thenature and/or degree of interaction between the subject molecule and anagent of interest. As detailed above, any suitable technique can beutilised. Such techniques would be known to the person of skill in theart and can be utilised in this regard. In terms of the nature of thesubject interaction, it may be desirable to assess the types ofinteractive mechanisms which occur between specific residues of anygiven agent and those of the molecule (for example, peptide bonding orformation of hydrogen bonds, ionic bonds, van der Waals forces, etc.)and/or their relative strengths. It may also be desirable to assess thedegree of interaction which occurs between an agent of interest and thesubject molecule. For example, by analysing the location of actual sitesof interaction between the subject agent and molecule it is possible todetermine the quality of fit of the agent into a region of the moleculeand the relative strength and stability of that binding interaction. Theform of association which occurs may not necessarily involve theformation of any chemical interactive bonding mechanism, as this istraditionally understood, but may involve a non-bonding mechanism suchas the proximal location of a region of the agent relative to a bindingregion of the molecule, for example, to effect steric hindrance withrespect to the binding of an activating molecule. Where the interactiontakes the form of hindrance or the creation of other repulsive forces,this should nevertheless be understood as a form of “interaction”despite the lack of formation of any of the traditional forms of bondingmechanisms.

It should also be understood that the molecule which is utilised eitherin a physical form or as an image, as hereinbefore discussed, to assessthe interactive complementarity of a putative agent may be a naturallyoccurring form of the molecule or it may be a derivative, homologue,analogue, mutant, fragment or equivalent thereof. The derivative,homologue, analogue, mutant, fragment or equivalent thereof may takeeither a physical or non-physical (such as an image) form.

The development of methodology for screening for GPI glycanimmunointeractive molecules in the context of the diagnosticapplications hereinbefore described facilitates determination of thethree dimensional structure of the immunointeractive molecule's bindingsite and the identification and/or rational modification and design ofagents which can interact with this site, for example, in the context ofanti-toxic vaccine development.

Without limiting the application of the present invention in any way,the method of the present invention facilitates the analysis, designand/or modification of agents capable of interacting with theseimmunointeractive molecules. In this regard, reference to “analysis,design and/or modification” of an agent should be understood in itsbroadest sense to include:

-   (i) Randomly screening (for example, utilising routine    high-throughput screening technology) to identify agents which    interact with the subject molecule. In this regard, crystals could    be soaked with said agents or co-crystalisation could be performed.    Such agents are often commonly referred to as “lead” agents in terms    of the random screening of proteinaceous or non-proteinaceous    molecules for their capacity to function in a desired manner.    Further, for example, binding affinity and specificity could be    enhanced by modifying lead agents to maximise interactions with the    molecule. Such analyses would facilitate the selection of agents    which are the most suitable for a given purpose. In this way, the    selection step is based not only on in vitro data but also on a    technical analysis of sites of agent: molecule interaction in terms    of their frequency, stability and suitability for a given purpose.    For example, such analysis may reveal that what appears to be an    acceptable in vitro activity in respect of a randomly identified    agent is in fact induced by a highly unstable interaction due to the    presence of proximally located agent: molecule sites which exhibit    significant repulsive forces thereby de-stabilising the overall    interaction between the agent and the molecule. This would then    facilitate the selection of another prospective lead compound,    exhibiting an equivalent degree of in vitro activity, but which    agent does not, upon further analysis, involve the existence of such    de-stabilising repulsive forces.-    Screening for the agents herein defined can be achieved by any one    of several suitable methods, including in silico methods, which    would be well known to those of skill in the art and which are, for    example, routinely used to randomly screen proteinaceous and    non-proteinaceous molecules for the purpose of identifying lead    compounds.-    These methods provide a mechanism for performing high throughput    screening of putative modulatory agents such as the proteinaceous or    non-proteinaceous agents comprising synthetic, recombinant, chemical    and natural libraries.-   (ii) The candidate or lead agent (for example, the agent identified    in accordance with the methodology described in relation to point    (i)) could be modified in order to maximise desired interactions    (for example, binding affinity to specificity) with the molecule and    to minimise undesirable interactions (such as repulsive or otherwise    de-stabilising interactions).-    Methods of modification of a candidate or lead agent in accordance    with the purpose as defined herein would be well known to those of    skill in the art. For example, a molecular replacement program such    as Amore (Navaza, J., (1994), Acta Cryst, A50:157-163) may be    utilised in this regard. The method of the present invention also    facilitates the mutagenesis of known signal inducing agents in order    to ablate or improve signalling activity.-   (iii) In addition to analysing fit and/or structurally modifying    existing molecules, the method of the present invention also    facilitates the rational design and synthesis of an agent, based on    theoretically modelling an agent exhibiting the desired interactive    structural features followed by the synthesis and testing of the    subject agent.

It should be understood that any one or more of applications (i)-(iii)above, may be utilised in identifying a particular agent.

The present invention also extends to the use of the molecules generatedin accordance with this aspect of the present invention in accordancewith the therapeutic, prophylactic and diagnostic methods hereinbeforedescribed.

The present invention is further described by the following non-limitingExamples.

Example 1 Reagents, Animals and Preparation of Parasites

Pronase was obtained from Boehringer Mannheim.Phosphatidylinositol-specific phospholipase C was from Calbiochem.Octyl-Sepharose, Protein-G Sepharose, n-octylthioglucopyranoside(n-otg), phenylmethylsulfonylfluoride (PMSF),p-tosyl-L-lysine-chloromethylketone (TLCK),N-tosyl-L-phenylalaninechloromethylketone (TLCK),p-chloro-mercuriphenylsulphonic acid (p-CMPS), aprotinin, leupeptin,pepstatin, iodoacetamide, n-ethyl-maleimide (NEM), and Concanavalin-Awere obtained from Sigma Chemical Co. Sephadex was from Pharmacia.Biogel P4 was from Biorad. Analytical or HPLC grade, acetic acid,butanol, chloroform, diethyl ether, ethanol, methanol and water wereobtained from BDH and Waters. Silica G60 TLC plates were from MerckDarmstadt. Tritiated mannose, glucosamine, myristic and palmitic acidswere from Amersham.

Adult female C57BL/6 and C3H/HeJ mice were bred and maintained in theWalter and Eliza Hall Institute specific pathogen free animal facility.

The FCB-1 line of Plasmodium falciparum were grown in vitro by standardmethods, and confirmed free of Mycoplasma contamination. For thebiosynthetic labelling of parasite proteins, 3H-palmitic acid conjugatedto defatted bovine serum albumin in molar ratio 1:1, 3H-glucosamine or3H-mannose were added at a final specific activity of 10 μCurie/ml, toRPMI 1640 cultures of 2×10¹⁰ parasites at the late trophozoite/earlyschizont stage for 2 hours (for labelling of GPI precursors) or 8 hours(for labelling of protein-bound GPI). Parasites were harvested by 0.05%Saponin lysis and centrifugation in the cold at 15,000 g for 20 minutes,followed by two washes in PBS and storage at −70° C.

Example 2 Purification of the 195 KD MSP-1 and 56 KD MSP-2 Antigens

The GPI-anchored MSP-1 and MSP-2 merozoite surface proteins werepurified to homogeneity as described previously (Schofield and Hackett(1993), supra). Biosynthetically labelled malaria parasites at the lateschizont stage were lysed in 0.05% Saponin and centrifuged at 15,000 gfor 20 minutes, and washed as above. The pellet was extracted in 25 mMn-octyl-thioglucopyranoside (n-otg), 1% BSA, 1 mM EDTA, 0.1M EGTA, 1 mMPMSF, 1 mM TLCK, 0.1 mM TLCK, 5 mM pCMPS, 1 μg/ml pepstatin, 1 μg/mlleupeptin, 1 mM NEM, 5 mM iodoacetamide, 150 mM NaCl, 25 mM Tris/HCl pH7.4 by sonication on ice. The extract was clarified by centrifugation at20,000 g for 30 minutes in the cold, and the supernatant decanted andloaded onto two immunoaffinity columns arranged in sequence, containingapproximately 10 mg monoclonal antibody 111.4 or monoclonal antibody113.1, each cross-linked to Protein G-Sepharose by gluteraldehyde (allprocedures on ice). The protein extract was passed through the column ata rate of 0.3 ml/min. The columns were washed first with 100 ml 10 mMn-otg, 1% BSA, 300 mM NaCl, followed by 100 ml 10 mM n-otg, 300 mM NaCl.Antigen was eluted from each column with four column volumes of 10 mMn-otg, 200 mM glycine pH 2.8. The pH of the eluate was neutralized with2M Tris. Aliquots of protein were analysed for purity by SDS-PAGEfollowed by staining with Coomassie brilliant blue. The remainingpurified proteins were dialysed exhaustively against 100 mM NH₄HCO₃using dialysis membrane previously boiled exhaustively in 10 mM EDTAfollowed by boiling in 10 changes of double distilled water. Proteinconcentration was determined by standard methods.

The remaining detergent soluble extract was made up to 1 mM CaCl₂, 1 mMMgCl₂, and 1 mM MnCl₂, and passed over a Con-A sepharose column,followed by washing with 10 column volumes of extraction buffer. Thecolumn was first eluted with detergent buffer containing 0.5Mα-methyl-mannopyranoside and 0.5M α-glucopyranoside, followed by 25 mMn-otg in 8M urea. Aliquots were subject to SDS-PAGE and fluorography orstaining with Coomassie blue.

Example 3 Purification of the C-Terminal GPI Anchors of Defined ParasiteAntigens

To purify the intact C-terminal GPIs free of detergent, non-covalentlybound lipids, glycolipids, phospholipids and protein or peptidefragments, affinity purified MSP-1 and MSP-2 were first scrubbed withorganic solvents. 10 mg/ml GPI-anchored proteins were placed in 150 μlaliquots in clean glass tubes. 600 μl MeOH was added and vortexed,followed by 150 μl 1 Chloroform and 450 μl water and further vortexing.The samples were centrifuged at 14000 rpm for 3 min, the supernatantdiscarded, and the interphase and lower phase mixed with 450 μl MeOH andre-centrifuged at 14000 rpm for 3 min. The protein pellet was extracted5 times with C/M/W 10:10:3, and finally extracted with acetone overnight at −20° C. The acetone was removed completely and the proteinstaken up with sonication in 6M Urea, 1 mM DTT, 1 mM iodoacetic acid.After 15 minutes at room temperature, the sample was diluted 6 fold andmade to 5 mM CaCl₂. 2.5% pre-digested Pronase B was added for 72 h at37° C. with 2 additions of 0.3% pronase. The digested sample was phaseseparated between water and water-saturated butanol, and the organicphase back extracted with water. The butanol phase was spotted onto TLCplates (Si-60) and run in the solvent system C/M/HAc/W 25:15:4:2. Thepronase-digested GPI fragment free of contaminants remains close to theorigin, and was detected by Berthold Digital Autoradiograph. Theappropriate region was scraped and the material eluted twice with C/M/W10:10:3 followed by 40% 1-propanol in water. The material was driedunder nitrogen gas, and once more separated between water andwater-saturated butanol.

Example 4 Purification of GIPLs and GPI Biosynthetic Precursors by TLC

GPI biosynthetic intermediates and non-protein bound mature GPI specieswere purified by TLC. 2×10¹⁰ P. falciparum schizonts were labelled with1 mCi ³[H]-mannose or ³[H]-palmitic acid in 250 ml glucose deficientRPMI 1640 supplemented with 40 mM fructose and 0.5% Albumax (GIBCO) for2 hours. Parasites were harvested by saponin lysis and washed twice inPBS. They were extracted three times in chloroform/methanol (2:1) andthree times in chloroform/methanol/water (1:1:0.3). Thechloroform/methanol extracts were subject to repeated Folch washing andthe chloroform phase dried in a Speedvac. The chloroform/methanol/waterextracts were dried down and partitioned between water andwater-saturated butanol. The butanol phase was washed with water anddried in a Speedvac. Both residues were then separated by TLC and theplates scanned by Bertold Digital Autoradiograph TLC scanner. Theradiolabelled peaks were identified and removed by scraping andre-extraction followed by drying. Areas lying between and outside theidentifiable peaks were treated in the same way, as were sham plates. Insome experiments, the GPIs were further purified over Octyl-Sepharose.Samples were taken up in 5% 1-propanol in 100 mM Ammonium acetate andloaded at a flow rate of 0.1 ml/min onto an Octyl-Sepharose column, andthe column washed with 100 mM Ammonium acetate, 5% 1-propanol. Thecolumn was eluted in a gradient running from 10 mM Ammonium acetate, 5%1-propanol to 60% 1-propanol in water. GPI containing fractions werelyophilised and flash evaporated in methanol.

Example 5 Generation of Chemical and Enzymatic Hydrolysis Fragments ofGPIs

Purified, glucosamine-labelled P. falciparum GPIs, in which all dpmswere detected in the organic phase following butanol/water partitioning,were subject to base hydrolysis by suspension in methanol/ammonia 1:1for 6 hours at 50° C., followed by partitioning between water and watersaturated butanol. Essentially 100% of label was then recovered from theaqueous phase. The aqueous phase was twice extracted withwater-saturated butanol, lyophilized, and flash evaporated withmethanol.

Example 6 DEAE Anion Exchange Chromatography

GPIs were loaded onto a A DEAE column in 99% methanol, 1% water andwashed with ten column volumes of solvent. They were subsequently elutedin 100 mM Ammonium Acetate in 99% methanol, 1% water and dried underNitrogen.

Example 7 Biogel P4 Size-Exclusion Chromatography

Ease-hydrolysed GPI glycans were spiked with phenol red and blue dextranin 10 mM Ammonium Acetate and further size-fractionated by passagethrough a 1 cm×1.2 meter Biogel P4 column equilibrated in 100 mMAmmonium acetate in water. The column had previously been exhaustivelycalibrated by repeated analytical runs with GPI mixed with acidhydrolyzed dextran markers to yield the relative elution position ofglucose units detected by staining with orcinol in concentrated sulfuricacid. The column runs proved to be highly reproducible. For preparativepurposes the dextran markers were omitted. The GPI peak was detected byscintillation counting of aliquots.

Example 8 Compositional Analysis by GC/MS

Glycan concentration and compositional purity was determined by GC-MS,following acid methanolysis and trimethylsilyl (TMS) derivatization.myo-Inositol content was measured following acid hydrolysis (6N HCl,110° C., 16 h) and TMS derivatization, with selected ion monitoring form/z 305 and 318. scyllo-Inositol was used as internal standardthroughout.

Example 9 Coupling of GPI Glycan to Maleimide-Activated KLH

The GPI glycan was exposed to 1 mM Traut's reagent (2-iminothiolane) in60 mM triethanolamine, 7 mM potassium phosphate, 100 mM NaCl, 1 mM EDTA,pH 8.0 in the cold for 90 minutes under nitrogen. The sample was thendesalted by gel filtration at 4° C. through a small Biogel P4 columnequilibrated in 7 mM potassium phosphate, 100M NaCl, 1 mM EDTA, pH 7.2and added to maleimide-activated KeyHole Limpet Haemocyanin (KLH) orOvalbumin (OVA) in coupling buffer (7 mM potassium phosphate, 100 mMNaCl, 1 mM EDTA, pH 7.2) overnight. The degree of conjugation wasestimated by comparison of cpms before and after dialysis of the sampleagainst PBS, or by use of Ellman's reagent for the quantitation ofsulfhydryl groups. Excess reactive sites were blocked with cysteine.

Example 10 Epitope Mapping of Anti-GPI Antibodies

Coupling of the purified GPI glycan to proteins was undertaken as above.To measure anti-lipid reactivities, we utilized commercially availablephosphatidylinositol from Sigma with identical composition to themalarial GPI, namely dipalmitoyl-PI. 2 mg PI was coupled to defatted BSAaccording to published protocols (Bate, C. A. W., Taverne, J.,Kwiatkowski, D., and Playfair, J. H. L. (1993) Immunology 79:138-145).

Example 11 ELISA Assay

Antigen (GPI-OVA, Glycan-OVA, BSA-PI, OVA or BSA alone) at 20 μg/ml inphosphate binding buffer was incubated overnight in 50 μl volumes inflat-bottomed Immunlon 96-well plates, followed by extensive washingwith buffer. The plates were blocked with 1% BSA, 1% OVA in PBS forseveral hours. From a 1/32 dilution, sera were titrated two-fold in 1%BSA, 1% OVA in PBS, and 501 aliquots incubated in triplicate for 2 hoursat room temperature, followed by extensive washing with 1% BSA, 1% OVA0.05% Tween-20 in PBS. An aliquot of affinity purified, biotin-labelledisotype specific goat anti-mouse second antibody was incubated as above,followed by further washing and the addition of streptavidin-alkalinephosphatase. After 30 minutes the plates were washed again andcolourimetric development initiated by the addition ofp-Nitrophenylphosphate in diethanolamine buffer. Background binding toBSA/OVA-coated plates was determined in parallel. The end-titres derivedare the last point giving values statistically different by two-wayanalysis of variance from non-specific binding by the same serum to theBSA/OVA-coated plates.

Example 12 Competition ELISA

From a 1/32 dilution, sera or mAbs were titrated two-fold in 1% BSA inPBS, 0.05% Tween-20, and pre-incubated for 4 hours at room temperaturewith a molar excess of competitior (20 μg/ml PI, or phosphatidylserine(PS), or diluent alone). Antigen (BSA-PI or BSA alone) at 20 μg/ml inphosphate binding buffer was incubated overnight in 50 μl volumes inflat-bottomed Immunlon 96-well plates, followed by extensive washingwith buffer. The plates were blocked with 1% BSA in PBS for severalhours. 50 μl aliquots of titrated antibody with or without competitorwere incubated in triplicate for 2 hours at room temperature, followedby extensive washing with 1% BSA 0.05% Tween-20 in PBS. An aliquot ofaffinity purified, biotin-labelled isotype specific goat anti-mousesecond antibody was incubated as above, followed by further washing andthe addition of streptavidin-alkaline phosphatase. After 30 minutes theplates were washed again and colourimetric development initiated by theaddition of p-Nitrophenylphosphate in diethanolamine buffer. Backgroundbinding to BSA-coated plates was determined in parallel. The end-titresderived are the last point giving values statistically different bytwo-way analysis of variance from non-specific binding by the same serumto the BSA-coated plates.

Example 13 Production of Monoclonal Antibodies

Monoclonal antibodies to the lipid domain of the GPI were produced aspreviously described (Tachado et al (1996) supra). Monoclonal antibodiesto the glycan were generated by immunization of OVA-TCR transgenic miceon a Balb/c background with OVA-glycan, followed by fusion and screeningof hybridoma culture supernatants against BSA vs. BSA-glycan.

Example 14 Macrophage Culture and TNF Output

LPS-nonresponsive C3H/HeJ macrophages were obtained as previouslydescribed (Schofield and Hackett (1993), supra and Tachado et al (1996)supra). 2×10⁵ adherent cells/well were given medium alone or testagents. 3 hrs after incubation TNF-α levels in the supernatant andstandard curve were determined by capture ELISA (Pharmingen).

Tyrosine Phosphorylation.

Rapid onset tyrosylphosphorylation was determined as previouslydescribed (Tachado et al (1997), supra).

Example 15 PI-PLC Treatment and FACS Analysis

2×10⁵ cells were exposed to 1 U/ml PI-PLC at 37° C. for 2 hours,followed by washing. They were then incubated in ice cold murinetonicity RPMI 1640 with 0.05% Sodium azide and 1% BSA with monoclonalantibodies or murine sera followed by washing and a further incubationwith isotype-specific FITC-conjugated antibody to mouse immunoglobulins.After washing in the same medium the cells were counter-stained with 0.5μg/ml propidium iodide and analysed by FACSscan.

Example 16 Immunization of Mice with Free GPI

Mice were immunized by three successive boosts of free intact malarialGPI emulsified in Incomplete Freund's Adjuvant spaced two weeks apart.Control mice received an equal amount of IFA alone. After immunization,sera were bled and the titres of anti-GPI antibodies determined byELISA. All animals immunized with GPI developed broadly similar levelsof anti-GPI antibodies (range 1/1024-1/4096) among individual animals.The anti-GPI response was predominantly IgM, and epitope mapping studiesby competition ELISA revealed that the antibody response was directedpredominantly towards the lipidic (phosphatidylinositol, PI) domain ofthe molecule, with some cross-reactivity to other phospholipiddeterminants (FIG. 1). Two weeks after the final boost mice werechallenged with P. berghei ANKA. Parasitaemia, the development ofneurological complications, and mortality were recorded daily. Nodifference in parasitaemia was observed. In the control group, 100% ofanimal manifested between day 5 and 9 an aggressive cerebral syndromewith neurological signs proceeding to rapid death with 12 hours. Inanimals immunized with intact free GPI, however, deaths occurred at anoticeable faster rate (FIG. 2).

The increased death rate in animals immunized with free GPI andsubsequently challenged with malaria may result from unanticipatedautoreactivity of anti-GPI antibodies. A panel of IgM monoclonalantibodies was derived from mice immunized with free GPIs. mAbs selectedat random from this panel were shown by PI-specific ELISA to be reactivewith PI domain of the molecule (FIG. 3), as was expected given theestablished serological specificity of the polyclonal sera of the donorimmunized mice (FIG. 1). In addition, these mAbs and the polyclonalantisera of GPI-immunized mice were shown by FACS analysis to react withhost GPI molecules expressed at the cell surface. Although surprising,the recognition of GPI-associated lipidic determinants at the cellssurface is not without precedence (Xia, M.-Q., Hale, G., Lifely, M. R.,Ferguson, M. A. J., Campbell, D., Packman, L., and Waldmann, H. (1993)Biochemical Journal 293:633-640). Pre-treatment of host cells withphosphatidylinositol-specific phospholipase C resulted in loss ofbinding of these mAbs, demonstrating formally that a lipidic moiety ofGPI molecules is exposed at the cell surface and is accessible forbinding by autoreactive antibodies generated in response to exposure tofree malarial GPI (FIG. 4). The binding was also shown to cause massiverapid onset intracellular tyrosine phosphorylation (FIG. 5), awell-known and predictable consequence of cross-linking host GPIs at thecell surface (Shenoy-Scaria, A. M., Kwong, J., Fujita, T., Olszowy, M.S., Shaw, A. S., and Lublin, D. M. (1992) Journal of Immunology149:3535-3541 and Stefanova, I., Corcoran, M. L., Horak, E. M., Wahl, L.M., Bolen, J. B., and Horak, I. D. (1993) Journal of BiologicalChemistry 268:20725-20728). Following binding of these antibodies tomacrophages, the cells responded more vigorously to stimulation withGPI, phorbol esters or malaria parasite extracts (FIG. 6). Upon passivetransfer into mice, these mAbs were sufficient to cause an increasedrate of death as compared with control IgM mAbs (FIG. 7).

Thus to summarize: (i) immunization of mice with the free P. falciparumGPI generates IgM reacting predominantly with the PI domain of the GPI;(ii) this immunization appears to exacerbate the P. berghei cerebralmalaria syndrome; (iii) exacerbated pathogenicity as detected byincreased death rate was also observed upon passive transfer of IgMmonoclonals with the same reactivity; (iv) the mAbs were shown tocross-react with host GPIs by FACS analysis, thereby causing massiveintracellular tyrosylphosphorylation and sensitization of macrophagesresulting in increased TNF output in response to addition agonists.Therefore it is proposed that a novel mechanism exists by which theacquisition of certain auto-reactive immunological specificities resultsin increased physiological sensitization to malarial toxins.

Example 17 Immunization of Mice with the GPI Glycan Conjugated to KLH

Previous publications dealing with the prospect of anti-disease vaccinesagainst malaria have proposed immunizing against a phospholipid domainwithin the putative toxin (Bate, C. A. W., Taverne, J., and Playfair, J.H. L. (1992c) Infection and Immunity 60:1894-1901, Bate, C. A., Taverne,J., Roman, E., Moreno, C., and Playfair, J. H. L. (1992a) Immunology75:129-135, Bate, C. A. W., Taverne, J., Bootsma, H. J., Mason, R. C. S.H., Skalko, N., Gregoriadis, G., and Playfair, J. H. L. (1992b)Immunology 76:35-41, Bate et al (1993) supra, Jakobsen, P. H.,Morris-Jones, S. D., Hviid, L., Theander, T. G., Hoier-Madsen, M.,Bayoumni, R., and Greenwood, B. M. (1993b) Immunology 79:653-657, Bate,C. A. W. and Kwiatkowski, D. (1994) Infection and Immunity 62:5261-5266and Playfair, J. H. L. (1994) Immunology Letters 43:83-86). The presentdata indicate strongly that this may be deleterious and should beavoided. It was sought to develop a novel approach, namely to detoxifyand deacylate the GPI and to determine whether immunization against theglycan domain of the molecule would exacerbate disease or be sufficientto protect mice against malarial pathology. Mice (n=7) were immunized bythree successive boosts of 50 μg KLH-glycan emulsified in IncompleteFreund's Adjuvant spaced two weeks apart. Two separate control groups(n=8 each) comprised animals receiving an equal amount of shamconjugated KLH in IFA, or those left untreated. After immunization, serawere bled and the titres of anti-GPI antibodies determined by ELISA. Allanimals immunized with KLH-glycan developed detectable anti-GPI glycanIgG antibodies, although there were differences in end-titre (range1/128-1/4096) among individual animals. The sera from vaccine recipients(but not sham-KLH controls) were able to inhibit TNF output frommacrophages stimulated with crude P. falciparum extracts, providingconvincing proof-of-principle for the neutralization of pathogenicity(FIG. 8). In contrast to the host-reactive antibodies to the GPI lipiddomain, pre-exposure of macrophages to these sera did not result inincreased TNF output in response to additional agonists. With these serait was not possible to detect significant cross-reactivity with hostGPIs at the cell surface as judged by FACS analysis of antibody bindingto host cells.

The P. berghei ANKA murine cerebral malaria model has many features incommon with the human cerebral malaria syndrome. It is a TNF-α andinterferon-γ (IFN-γ) dependent encephalitis associated with upregulationof ICAM-1 on the cerebral microvascular endothelium, an increase in bothparasite and macrophage/neutrophil adherence to these target cells, andattendant neurological complications. Unlike human cerebral malaria,there is a breakdown of the blood-brain barrier in the terminal stagesof the murine syndrome. However, in the proximal stages the murinedisease reflects more accurately the inflammatory cascade leading tocerebral involvement in humans. To determine whether anti-GPIimmunization prevents cerebral pathogenesis in vivo, mice were immunizedwith P. falciparum IPG conjugated to KLH. Two weeks after the finalboost mice were challenged with P. berghei ANKA. Parasitaemia, thedevelopment of neurological complications, and mortality were recordeddaily. No difference in parasitaemia was observed among groups. In bothcontrol groups, 87.5% of animal manifested between day 7 and 12 anaggressive cerebral syndrome with neurological signs proceeding to rapiddeath with 12 hours, and 12.5% did not develop the syndrome. As therewere no significant differences between sham-immunized and untreatedgroups, the data from these two control groups are pooled (FIG. 9). Inrecipients of KLH-glycan, one animal (14.2%) died with similar kinetics,two animals (28.5%) developed the cerebral syndrome with substantiallydelayed kinetics (on days 10 and 11, and showing prolonged course ofsyndrome before succumbing), and four animals (57.2%) were completelyprotected, failing to develop the cerebral syndrome at any stage (FIG.9). Thus immunization of mice with the P. falciparum GPI glycancovalently linked to a carrier protein affords substantial protectionagainst the P. berghei cerebral malaria syndrome.

A panel of monoclonal antibodies was made from mice immunized withpurified GPI glycan conjugated to OVA (OVA-glycan). The hybridoma fusionproducts were initially screened for binding to BSA-glycan as comparedto BSA alone. Over 80 glycan-reactive IgG monoclonal antibodies weredetected. Of these, many were reactive with parasites but not hosterythrocytes as judged by the Indirect Fluorescent Antibody Test.Purified monoclonal antibodies 1G7 and 3G6 were sufficient to block theinduction of TNF by 100% when added at low concentration to total crudeparasite extracts (FIG. 10). To determine whether anti-GPI antibodiesalone are sufficient to prevent severe malarial pathology, mice wereinfected with 10⁶ P. berghei ANKA i.p. On day 4 they were divided atrandom into 10 controls receiving an irrelevant IgG and groups of 5receiving mAbs 1D12, 2C4, 3G5 and 4C3 raised against the P. falciparumGPI inositolphosphoglycan. All mice received 100 μg antibody/day i.p.for 7 days. Mice were monitored for parasitaemia daily and clinicalsigns every 6 hours. 100% of controls died of the cerebral malariasyndrome between days 6 and 8 post-infection. Throughout this period, noanimals receiving either of the 4 anti-GPI monoclonal antibodies showedsigns of illness, despite being equally parasitized as controls. On day10 one of the 5 animals receiving monoclonal 3G5 died. Other than thisindividual, no others showed cerebral signs and none died (FIG. 11).Thus 19/20 (95%) of the 20 animals receiving anti-GPI mAbs survived, vs.zero survival in controls (n=30 total). Parasitaemias were identical intest and control groups throughout the experiment. For visual clarity,the figure shows the 4 treatment groups in aggregate. In addition, 5mice received antibodies alone without parasite challenge. There were nodetectable acute or toxic reactions in these mice receiving antibodiesalone.

In addition a standard murine model of TNF-driven lethality was used todetermine whether GPI mediates parasite-induced acute toxic shock. Thismodel manifests disseminated intravascular coagulation, peripheralvascular failure and shock, and thus has clinical features in commonwith the human “algid malaria” syndrome. LPS-non-responsive C3H/HeJ andC57B16 mice were primed with 20 mg D-galactosamine followed after 1 hourby purified GPI, PE or PBS alone. Mice receiving D-galactosaminefollowed by vehicle alone showed 100% survival. Both PE and purified GPIinduced lethal shock in 100% of D-galactosamine-primed C3H/HeJ andC57B16 recipients. mAbs to the GPI glycan substantially preventedTNF-driven lethality in vivo (FIG. 12).

Example 18 Synthetic GPI as a Candidate Anti-Toxic Vaccine AgainstMalaria

Methods

Protein/Glycan Conjugation

Synthetic GPI glycan 1 was reacted with 10-fold molar excess of Traut'sreagent (2-iminothiolane) in 60 mM triethanolamine, 7 mM potassiumphosphate, 100 mM NaCl, 1 mM EDTA, pH 8.0 in the cold for 90 minutesunder nitrogen, to introduce a sulfhydryl onto the free primary amine(ethanolamine). The sample was desalted by Giogel P4 filtration incoupling buffer at 4° C., and the sample added to maleimide-activatedKLH or OVA (Pierce) overnight. After exhaustive dialysis against water,conjugation efficiency was estimated by Gas Chromatrography/MassSpectroscopy. Samples were hydrolysed in 6N HCl and the trimethylsilylderivatives quantified for myo-inositol content by selective ionmonitoring, using scyllo-inositol as internal standard. For thegeneration of sham-conjugated carrier proteins, maleimide-activated KLHor OVA (Pierce) were subjected to identical procedures except cysteinewas substituted for sulfhydryl-modified glycan.

Infections

All experiments were in accordance with local Animal Ethics Committeeregulations. Young adult C57BL6 mice from Jackson Laboratories werepre-bled and inoculated with 6.5 μg KLH-glycan (0.176 μg glycan, n=16)or KLH-cysteine (sham-immunized, n=24) emulsified in Freund's CompleteAdjuvant, and boosted with equal amounts of immunogen in IncompleteFreund's Adjuvant at 2 weekly intervals. After two boosts they wererested and injected i.p with 1×10⁶ P. berghei ANKA-infectederythrocytes. Naïve mice (n=12) served as unimmunized controls.Parasitemias were assessed from Giemsa-stained thin films. Mortality waschecked twice daily. Mice were judged as developing cerebral malaria ifdisplaying neurological signs such as loss of reflex or ataxia, anddying between days 5 to 12 post-infection with relatively lowparasitemia levels (below 15%). Differences in survival curves of P.berghei infected mice across this time-period were assessed byCox-Mantel logrank transformation on Kaplan-Meier plots. Deaths from day14 onwards with high parasitaemia (>60%) and low rates of cerebralvascular occlusion were ascribed to hyperparasitaema/haemolysis, and allanimals failing to develop cerebral malaria eventually succumbed to thissyndrome.

Pathology

For histological analysis of cerebral pathology brains were taken into10% neutral-buffered formalin, sectioned (5μ) and stained withHaemotoxylin/Eosin. In other experiments, groups of 6 naïve,sham-immunized and KLH-glycan immunized mice were challenged as above.All mice were sacrificed at day 6, along with age/sex matched uninfectedcontrols, their serum collected for determination of pH, and lungsremoved. The wet weight was determined immediately after removal of theorgan, and the dry weight after overnight incubation at 80° C. (25).Brains were taken for histological examination as above.

TNF Output

Mycoplasma-free P. falciparum schizonts (3D7 strain) were prepared bygelatin flotation followed either by extraction with sample buffer (forSDS-PAGE and Western blots) or by saponin lysis and three washes inisotonic buffer. Parasites were taken up by sonication in completemedium and aliquots of 5×10⁶ cell equivalents in 100 μl volumespre-incubated for 1 hour with the indicated concentration of test orcontrol sera, followed by addition to 4×10⁵ target RAW264.7 cells for 16hours in a 96-well plate. Levels of TNFα in culture supernatants weredetermined by capture ELISA according to manufacturer's protocol(Pharmingen, San Diego, USA) and quantified by interpolation against arecombinant protein standard curves.

Immunofluoresence

Thin films of mature P. falciparum cultures at 10% parasitaemia werefixed in acetone at −20° C. and exposed to test and control antisera(1/80) followed after washing in PBS by 1/200 dilution offluorochrome-conjugated goat anti-mouse IgG (γ-chain specific). Slideswere photographed under appropriate illumination.

Statistics

Statistical comparison between test and control groups was by Student'st-test except for Kaplan-Meier survival plots, tested by Cox-Mantellogrank transformation.

Results

P. falciparum shows uniquely low levels of N- and O-linked glycosylation(Dieckmann-Schuppert, A., Bender, S., Odenthal-Schnittler, M., Bause, E.and Schwarz, R. T. (1992) Eur. J. Biochem. 205:815-825;Dieckmann-Schuppert, A., Bause, E. and Schwarz, R. T. (1993) Eur. J.Biochem. 216:779-788), and GPI accordingly represents at least 95% ofthe post-translational carbohydrate modification of parasite proteins(Gowda, D. C., Gupta, P. and Davidson, E. A. (1997) J. Biol. Chem.272:6428-6439). GPI structure is conserved across all parasite isolatesexamined to date (Berhe, S., Schofield, L., Schwarz, R. T. and Gerold,P. (1999) Mol. Biochem. Parasitol. 103:273-278). The bioactivity of GPIsagainst target host tissues requires the contribution of both lipid andcarbohydrate domains, and deacylation of GPIs by enzymatic or chemicalhydrolysis renders the carbohydrate moiety non-toxic (Schofield et al.(1993), supra; Tachado, S. D., and Schofield, L. (1994) Biochem.Biophys. Res. Commun. 205:984-991; Schofield et al. (1996), supra;Tachado et al. (1996), supra; Tachado et al. (1997), supra). Based onthe sequence of the non-toxic P. falciparum GPI glycan (Gerold, P.,Dieckmann-Schuppert, A. and Schwarz, R. T. (1994) J. Biol. Chem.269:2597-2606), the structureNH₂—CH₂—CH₂—PO₄-(Manα1-2)6Manα1-2Manα1-6Manα1-4GlcNH₂α1-6myo-Insositol-1,2cyclic phosphate was chemically synthesised (FIG. 13). Confirmation ofstructure was by MALDI-TOF mass spectrometry and ³¹P-NMR (D₂O). Toprepare an immunogen, the synthetic GPI glycan was treated with2-iminothiolane to introduce a sulfhydryl at the primary amine withinthe ethanolamine phosphate, desalted, and conjugated tomaleimide-activated Ovalbumin (OVA, in molar ratio 3.2:1) or Key-HoleLimpet Haemocyanin (KLH, in molar ratio 191:1). This material was usedto immunize mice.

The synthetic malarial GPI glycan was immunogenic in rodents. Antibodiesfrom KLH-glycan immunized animals gave positive IgG titres againstOVA-glycan but no sham-conjugated OVA-cysteine containing identicalcarrier and sulfhydryl bridging groups. No reactivity to GPI glycan wasdetected in pre-immune sera or in animals receiving sham-conjugated KLH.More significantly, antibodies raised against synthetic P. falciparumGPI glycan bound to native GPI as judged by several methods. These IgGantibodies recognized intact trophozoites and schizonts byimmunofluorescence (FIG. 14 a), with a subcellular distributionsuggestive of exclusion from merozoite nuclei. Pre-immune andsham-immunized sera failed to react. Anti-GPI failed to bind touninfected erythrocytes, despite these cells expressing endogenous GPIsof host origin (FIG. 14 a). However, in contrast to malaria GPI, allmammalian GPIs characterized to date show amino-sugar orphosphoethanolamine modifications to the core glycan structure(McConville et al. (1993), supra) and these epitopic differences mayaccount for the lack of cross-reactivity. As expected, anti-GPI glycanIgG detected multiple molecular species in Western blotting against P.falciparum-infected erythrocytes but not uninfected erythrocytes (FIG.14 b), consistent with the presence in mature schizonts of multipleGPI-modified proteins such as MSP-1, MSP-2, MSP4, MSP-5 as well as theirprocessing products. These results indicate that protein-specificfeatures do not greatly influence the binding of anti-glycan antibodiesto native GPI anchors. Structurally identical, non-protein linked freeGPIs occur in molar ratio 4:1 to protein-linked forms, and a frequentobservation was strong reactivity with these moieties running at thedye-front (FIG. 14 b). Pre-immune and sham-immunized sera failed toreact in Western blots (FIG. 14 b).

TNFα production by macrophages is widely used as a biochemical marker ofmalaria endotoxin activity in vitro. Purified malarial GPI is sufficientfor TNF production (Schofield et al. (1993) supra; Tachado et al. (1994)supra; Schofield et al. (1996) supra; Tachado et al. (1996) supra;Tachado et al. (1997), supra), but it remains unclear whether this agentis predominantly responsible for this activity in parasites. It wassought to determine whether antibodies specific for the P. falciparumGPI could neutralize parasite endotoxic activity in vitro, and toquantify the contribution of GPI to the total endotoxic activity ofmalaria. In contrast to pre-immune sera or those drawn fromsham-conjugated KLH-immunized controls, antibodies from mice immunizedwith KLH-glycan specifically neutralized TNFα output from macrophagesinduced by crude total extracts of P. falciparum (FIG. 14 c). Theseantibodies have no effect on macrophage viability or production of TNFαin response to unrelated agonists such as lipopolysaccharide. Thus GPIis required for the induction by malaria parasites of hostpro-inflammatory responses in vitro.

The murine P. berghei ANKA severe malaria model has salient features incommon with the human severe and cerebral malaria syndromes. It is aTNFα-dependent encephalopathy associated with upregulation of ICAM-1 onthe cerebral microvascular endothelium and attendant neurologicalcomplications (Grau, G. E., (1987) Science 237:1210-1212; Grau, G. E. etal. (1989) Proc. Natl. Acad. Sci. USA 86:5572-5574; Grau, G. E. et al.(1991) Eur. J. Immunol. 21:2265-2267; Jennings, V. M., Actor, J. K.,Lai, A. A. & Hunter, R. L. (1997) Infect. Immun. 65:4883-4887).Pulmonary oedema and lactic acidosis are also observed. Unlike most, butnot all, human cerebral malaria cases, the blood-brain barrier iscompromised in the terminal or agonal stages of the murine syndrome. Inthe proximal or developmental stages however the murine disease reflectsmore accurately the cytokine-dependent inflammatory cascade leading tocerebral involvement in humans. Thus early stage P. berghei ANKAinfection appears the best available small animal model of clinicallysevere malaria. To determine whether anti-GPI immunization preventssystemic and cerebral pathogenesis in this pre-clinical model, C57B16mice primed and twice boosted with 6.5 μg KLH-glycan (0.18 μg glycan) orKLH-cysteine in Freund's Adjuvant were challenged with Plasmodiumberghei ANKA, and the course of disease monitored. 100% of bothsham-immunized and naïve control mice died with the cerebral syndrome,showing severe neurological signs including loss of self-rightingreflex, ataxia, and hemiplegia, with pronounced hypothermia andoccasional haematouria (FIG. 15 a). These fatalities were evident earlyduring infection (day 5-8) with relatively low parasitaemias. There wereno differences between naïve and sham-immunized mice indicating exposureto carrier protein alone in Freund's Adjuvant does not influence diseaserates. In contrast, mice immunized with chemically synthetic P.falciparum GPI glycan coupled to KLH were substantially protectedagainst cerebral malaria, with significantly reduced death rates (75%survival, p<0.02, FIG. 15 a). In four separate additional experiments,results over the range of 58.3-75% survival over this time period invaccine recipients (n=50 total) vs. 0-8.7% survival in sham-immunizedcontrols (n=85) were obtained. Parasitaemias were not significantlydifferent between test and control groups in these experiments,demonstrating that prevention of fatality by anti-GPI vaccination doesnot operate through effects on parasite replication (FIG. 15 b). Thediagnoses of cerebral malaria, or absence of this condition, wereconfirmed by histological examination of brains taken at day 6post-infection. Sham-immunized mice showed typical pathology includinghigh levels of vascular occlusion with both parasitized RBCs and hostleukocytes (FIG. 15 c). Immunized animals in contrast showed absent ormuch reduced vascular occlusion despite similar parasite burdens (FIG.15 c).

Severe malaria in both humans and rodents may be associated withadditional organ-specific and systemic derangements, including pulmonaryoedema and serum acidosis. Acidosis may be a prime pathophysiologicalprocess and is the strongest single prognostic indicator of outcome. Thebiochemical aetiology of acidosis is unclear, and may result fromseveral causes including a metabolic consequence of cytokine excess,lactate production by parasites, decrease hepatic clearance of lactate,tissue hypoxia and respiratory insufficiency. The relationship to humanmalarial acidosis of that in the rodent model also remains to beelucidated. Nonetheless, it was sought to determine whether anti-GPIvaccination protects against these additional non-cerebral diseasesyndromes in mice. Both sham-immunized and naïve individuals developedpronounced pulmonary oedema by day 6 post-infection, as measured by lungdry:wet weight ratios, and this was markedly reduced in vaccinerecipients (FIG. 15 d). Similarly, whereas sham-immunized andunimmunized mice developed significant acidosis as shown by reducedblood pH at day 6 post-infection, in vaccinated mice blood pH wasmaintained at physiological levels (FIG. 15 e). As parasite burdens weresimilar in test and control groups, production of lactic acid byparasite biomass, and any mild haemolytic anaemia consequence uponparasitaemia at this stage, are not major contributors to acidosis inthis model. Clearly however, immunizing against GPI prevents thedevelopment of pulmonary oedema and acidosis as well as cerebral malariain P. berghei infection.

The experiments undertaken for this study were designed to test thehypothesis that GPI is implicated in the malarial inflammatory cascadeand in metabolic derangement, and to determine whether vaccinationagainst this target affords clinical protection in the best small animalmodel available. Mice were primed and boosted with 176 ng glycan perdose, which may be a sub-optimal quantity. Systematic optimization withrespect to formulation, carrier/hapten ratios, adjuvants, dosage ortiming of the immunization regimen would be a matter of routineprocedure to the person of skill in the art. Therefore it is possiblethat the degree of protection against disease observed here may beimproved further pursuant to such routine optimisation. Similarly,anti-GPI vaccination may conceivably be beneficial in other malarialdisease syndromes not sufficiently modeled by acute P. berghei ANKAinfection.

In areas of high transmission, immunity to malaria is acquired in twostages: after two to three years of susceptibility to severe and lethaldisease, children developed an acquired clinical immunity which protectsagainst life-threatening pathology despite persistent high parasitaemias(Christophers, S. R. (1924) Ind. J. Med. Res. 12:273-294; Sinton, J. A.(1939) Journal of the Malaria Institute of India 2:71-83; McGregor, I.A., Gilles, H. M., Walters, J. H., Davies, A. H. and Pearson, F. A.(1956) Br. Med. J 2:686-692). The chemical synthesis of GPI fragmentsreported here should aid in serological investigations and the epitopemapping of human anti-GPI antibodies.

In contrast to acquired clinical immunity, anti-parasite immunity takesmany more years to develop (McGregor et al (1956), supra) and is easilylost, reflecting the problems of antigenic diversity, antigenicvariation, redundancy in invasion pathways, immune evasion strategiesand problems of MHC-linked genetic restriction in the immune response toparasite antigens. Current approaches to anti-malarial vaccines seek toinduce anti-parasite immunity through parasiticidal mechanisms targetedto parasite protein antigens. The public health potential ofanti-disease vaccines is demonstrated by the highly effective tetanusand diphtheria toxoid vaccines which protect against the most injuriousconsequences of infection by targeting bacterial toxins (Schofield, F.(1986) Rev. Infect. Dis. 8:144-156). The findings of this studydemonstrate that GPI is the dominant endotoxin of malaria parasiteorigin. A non-toxic GPI oligosaccharide coupled to carrier protein isimmunogenic and provides significant protection against malarialpathogenesis and fatalities in a preclinical rodent model. It istherefore possible that GPI contributes to life-threatening disease inhuman malaria. These data suggest that an anti-toxic vaccine againstmalaria might be feasible and that synthetic fragments of the P.falciparum GPI may be developed further to that end.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more said steps or features.

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1. A method of eliciting or inducing, in a mammal, an immune responsedirected to a parasite said method comprising administering to saidmammal an effective amount of an immunogenic composition, whichcomposition comprises the inositolglycan domain portion of GPI, whichinositolglycan domain portion comprises insufficient lipidic domain toinduce or elicit an immune response directed to said lipidic domain andwhich has a terminal inositol-phosphoglycerol substituted with apositively or negatively charged moiety.
 2. A method of therapeuticallyor prophylactically treating a mammal for a parasite infection saidmethod comprising administering to said mammal an effective amount of animmunogenic composition which composition comprises the inositolglycandomain portion of GPI, which inositolglycan domain portion comprisesinsufficient lipidic domain to induce or elicit an immune responsedirected to a GPI lipidic domain and which has a terminalinositol-phosphoglycerol substituted with a positively or negativelycharged moiety.
 3. A method for the treatment and/or prophylaxis of amammalian disease condition caused by a parasite infection, said methodcomprising administering to said mammal an effective amount of animmunogenic composition which composition comprises the inositolglycandomain portion of GPI, which inositolglycan domain portion comprisesinsufficient lipidic domain to induce or elicit an immune responsedirected to said lipidic domain and which has a terminalinositol-phosphoglycerol substituted with a positively or negativelycharged moiety.
 4. The method according to claim 1, 2 or 3 wherein saidparasite is Plasmodium.
 5. The method according to claim 4 wherein saidPlasmodium is Plasmodium falciparum.
 6. The method according to claim 5wherein said GPI molecule is a Plasmodium falciparum GPI inositolglycandomain.
 7. The method according to claim 6 wherein said GPIinositolglycan domain is synthetically generated.
 8. The methodaccording to claim 7 wherein said GPI inositolglycan domain comprisesthe structure EtN-P-(Manα1,2)-6Mα1, 2Mα1, 6Manα1,4GlcNH₂α1-myo-inositol-1,2 cyclic-phosphate wherein EtN is ethanolamine,P is phosphate and M is mannose.
 9. The method according to claim 7wherein said GPI inositolglycan domain comprises the structureNH₂—CH₂—CH₂—PO₄-(Manα1-2) 6Manα1-2Manα1-6Manα1-4GlcNH₂-6myo-inositol-1,2 cyclic-phosphate.
 10. The methodaccording to claim 3 wherein said disease condition is malaria.
 11. Acomposition capable of inducing an immune response directed to aparasite said composition comprising a parasite GPI inositolglycandomain portion but which portion is incapable of inducing an immuneresponse to a lipidic domain of a GPI and which has a terminalinositol-phosphoglycerol substituted with a positively or negativelycharged moiety.
 12. A vaccine composition for inducing an immuneresponse to a parasite, said composition comprising as the activecomponent the parasite inositolglycan domain portion of GPI, whichinositolglycan portion is incapable of inducing an immune responsedirected to a lipidic domain of a GPI and which has a terminalinositol-phosphoglycerol substituted with a positively or negativelycharged moiety, together with one or more pharmaceutically acceptablecarriers and/or diluents.
 13. A pharmaceutical composition comprising aparasite GPI inositolglycan domain portion but which portion isincapable of inducing an immune response directed to a lipidic domain ofa GPI and which has a terminal inositol-phosphoglycerol substituted witha positively or negatively charged moiety, together with one or morepharmaceutically acceptable carriers and/or diluents.
 14. Thecomposition according to claim 11, 12 or 13 wherein said parasite isPlasmodium.
 15. The composition according to claim 14 wherein said GPIinositolglycan domain is synthetically generated.
 16. The compositionaccording to claim 15 wherein said synthetic GPI inositolglycan domaincomprises the structure EtN-P-(Manα1,2)-6Mα1, 2Mα1, 6Manα1,4GlcNH₂α1-myo-inositol-1,2 cyclic-phosphate, wherein EtN isethanolamine, P is phosphate and M is mannose.
 17. The compositionaccording to claim 16 wherein said GPI inositolglycan domain comprisesthe structure NH₂—CH₂—CH₂—PO₄-(Manα1-2)6Manα1-2Manα1-6Manα1-4GlcNH₂-6myo-inositol-1,2 cyclic-phosphate.
 18. Amethod for detecting, in a biological sample, an immunointeractivemolecule directed to a microorganism, said method comprising contactingsaid biological sample with a molecule comprising a modified GPIinositolglycan domain and qualitatively and/or quantitatively screeningfor said GPI inositolglycan domain-immunointeractive molecule complexformation.
 19. A method for detecting or monitoring an immune responsedirected to a microorganism in a subject said method comprisingcontacting a biological sample, from said subject, with a moleculecomprising a modified GPI inositolglycan domain which comprisesinsufficient lipidic domain to induce or elicit an immune responsedirected to a GPI lipidic domain and which a terminal inositolphosphoglycerol substituted with a positively or negatively chargedmoiety and qualitatively and/or quantitatively screening for GPIinositolglycan domain-immunointeractive molecule complex formation. 20.The method according to claim 18 or 19 wherein said modified GPImolecule is the inositolglycan domain portion of GPI.
 21. The methodaccording to claim 20 wherein said modified GPI molecule is a modifiedparasite GPI molecule.
 22. The method according to claim 21 wherein saidparasite is Plasmodium.
 23. The method according to claim 22 whereinsaid Plasmodium is Plasmodium falciparum.
 24. The method according toclaim 23 wherein said modified Plasmodium falciparum GPI molecule is aPlasmodium falciparum GPI inositolglycan domain.
 25. The methodaccording to claim 24 wherein said GPI inositolglycan domain issynthetically generated.
 26. The method according to claim 25 whereinsaid synthetic GPI inositolglycan domain comprises the structureEtN-P-(Manα1,2)-6Mα1, 2Mα1, 6Manα1, 4GlcNH₂α1-myo-inositol-1,2cyclic-phosphate, wherein EtN is ethanolamine, P is phosphate and M ismannose.
 27. The method according to claim 26 wherein said synthetic GPIinositolglycan domain comprises the structureNH₂—CH₂—CH₂—PO₄-(Manα1,2)6Mα1, 2Mα1, 6Manα1, 4GlcNH₂α1-myo-inositol-1,2cyclic-phosphate.
 28. The method or composition according to any one ofclaims 1, 2, 3, 11, 12 or 13 wherein the positively or negativelycharged moiety is a hydrophilic moiety.
 29. The method according to anyone of claims 1, 2, 3, 11, 12 or 13 wherein the positively or negativelymoiety comprises a phosphate moiety.
 30. The method according to any oneof claims 1, 2, 3, 11, 12 or 13 wherein the positively or negativelymoiety is inositol-1,2-cyclic phosphate.
 31. The method according toclaim 18 or 19 wherein the positively or negatively charged moiety is ahydrophilic moiety.
 32. The method according to claim 18 or 19 whereinthe positively or negatively charged moiety comprises a phosphatemoiety.
 33. The method according to claim 18 or 19 wherein thepositively or negatively charged moiety is inositol-1,2-cyclicphosphate.
 34. A modular kit comprising one or more members, wherein atleast one member is a solid support comprising a GPI molecule whichconsists of the Plasmodium falciparum GPI inositolglycan domain.
 35. Amodular kit comprising one or more members, wherein at least one memberis a solid support comprising the inositolglycan domain portion of GPI,which inositolglycan domain portion comprises insufficient lipidicdomain to induce or elicit an immune response directed to said lipidicdomain and which has a terminal inositol-phosphoglycerol substitutedwith a positively or negatively charged moiety.
 36. The modular kit ofclaim 35, wherein the positively or negatively charged moiety is ahydrophilic moiety.
 37. The modular kit of claim 35, wherein thepositively or negatively moiety comprises a phosphate moiety.
 38. Themodular kit of claim 35, wherein the positively or negatively moiety isinositol-1,2-cyclic phosphate.
 39. The method of claim 1, wherein saidparasite is Plasmodium.
 40. The method of claim 2, wherein said parasiteis Plasmodium.